Non-selective kinase inhibitors

ABSTRACT

Disclosed herein are compounds, compositions, and methods for preventing and treating proliferative diseases associated with aberrant receptor tyrosine kinase (RTK) activity. The therapeutic indications described herein more specifically relate to the non-selective inhibition of RTKs associated with vascular and pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/760,139 filed Jul. 9, 2015, which claims priority to National StageApplication of International Application No. PCT/US14/10778 filed Jan.9, 2014, which claims priority to U.S. Provisional Application No.61/751,217 filed Jan. 10, 2013, and U.S. Provisional Application No.61/889,887, the entire contents of which are hereby incorporated byreference in their entirety.

STATEMENT OF GOVERNMENT-SPONSORED RESEARCH

This invention was made with United States government support underGrant Number 1R43HL102946-01 and 2R44HL102946-02 awarded by the NationalInstitute of Health. The United States government has certain rights inthe invention.

FIELD OF THE INVENTION

The present disclosure relates generally to the treatment and preventionof disease associated with protein kinase activity. In particular, thepresent technology relates to therapeutic indications of protein kinaseinhibitors and methods for the treatment or prevention of pulmonary andvascular conditions, cancer, and other disorders.

BACKGROUND OF THE INVENTION

The following discussion of the background is merely provided to aid thereader in understanding the invention and does not necessarily describeor constitute prior art.

Receptor tyrosine kinases (RTKs) are transmembrane polypeptides thatregulate the regeneration, remodeling, development and differentiationof cells and tissues. See, e.g., Mustonen et al., J. Cell Biology 129,895-898 (1995); van der Geer et al. Ann Rev. Cell Biol. 10, 251-337(1994). In addition to activating RTKs, polypeptide ligand growthfactors and cytokines are capable of inducing conformation changes inRTK external domains which results in receptor dimerization.Lymboussaki, Dissertation, Univ. of Helsinki, Mol./Cancer Bio Lab andDept. of Pathology, Haartman Institute (1999); Ullrich et al., Cell 61,203-212 (1990). Cognate RTK receptor-ligand binding, moreover, impartsreceptor trans-phosphorylation at specific tyrosine residues andsubsequent activation of the kinase catalytic domains, thereby enablingsubstrate phosphorylation and activation of associated signalingcascades. Id.

Aberrant RTK activity, however, is associated with a variety of diseaseconditions and systemic delivery of certain RTK inhibitors have shownefficacy for specific disease conditions. In vivo assays to this end,including the murine monocrotaline (MCT) model system, have beenemployed for ascertaining whether putative RTK inhibitors would functionas therapeutic agents. Concerning preclinical drug candidate efficacy,however, the MCT model has been criticized inasmuch as such a systemfails to substantiate certain human disease phenotypes, e.g., thedevelopment of neointimal and/or plexiform lesions that aresymptomatically comorbid with such diseases. Hence, this model is animperfect system, which may confound the etiological and pathologicalindications of human disease. Thus, new or complementary model systemsare necessary for accurate and efficient drug development.

In concert with the development and administration of first generationRTK inhibitors, e.g., imatinib, RTKs have evolved inhibitor resistanceby acquiring certain mutations. See, e.g., Shah et al., Science, 305,395-402 (2004). For example, in diseased patients refractory to certainkinase inhibitors, e.g., imatinib, it has been shown that thehydrophobic pocket “gatekeeper residue” frequently possesses mutations.See Pao et al., PLos Med. 2(3):e73 (2005). Such mutations have beenidentified with respect to ABL, i.e., at the T315 residue, and atanalogous positions in KIT, PDGFRα, EGFR, and other kinases. Id. Hence,new RTK inhibitors with superior efficacy-developed in model systemsthat phenotypically resemble human disease pathology—are required forpreventing and treating diseases possessing aberrant RTK activity.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method of non-selectivekinase receptor inhibition for treating pulmonary disorders in asubject, including: administering to the subject a therapeuticallyeffective amount of a compound of Structure 1, a tautomer, enantiomer,isomer or stereoisomer of the compound, a pharmaceutically acceptablesalt of the compound, tautomer, enantiomer, isomer or stereoisomer ofthe compound, or any mixtures thereof, where Structure 1 has theformula:

And where X is independently selected from C, N, O, S or —CN;

R¹, R², and R³ may be the same or different and are independentlyselected from the group consisting of H, C, N, O, S, Cl, Br, F, I, —CN,—NO₂, —OH, —CH₃, —CF₃, —C—N—C— groups, —C—N—C(═O)— groups, —C(═O)R⁸groups, —N—C(═O)R⁸ groups, —C—N—C(═O)R⁸ groups, substituted andunsubstituted R⁸ groups, substituted and unsubstituted R⁸ groupssubstituted with one or more of R⁹, R¹⁰, and R¹¹, substituted andunsubstituted amidinyl groups, substituted and unsubstituted guanidinylgroups, substituted and unsubstituted primary, secondary, and tertiaryalkyl groups, substituted and unsubstituted aryl groups, substituted andunsubstituted alkenyl groups, substituted and unsubstituted alkynylgroups, substituted and unsubstituted heterocyclyl groups, substitutedand unsubstituted aminoalkyl groups, substituted and unsubstitutedalkylaminoalkyl groups, substituted and unsubstituted dialkylaminoalkylgroups, substituted and unsubstituted arylaminoalkyl groups, substitutedand unsubstituted diarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstitutedheterocyclylalkyl groups, substituted and unsubstituted cyano groups,substituted and unsubstituted pyrimidinyl groups, substituted andunsubstituted cyano(aryl) groups, substituted and unsubstitutedcyano(heterocyclyl) groups, and substituted and unsubstitutedcyano-pyrimidinyl groups;

R⁴, R⁵, R⁶, and R⁷, may be the same or different and are independentlyselected from the group consisting of H, Cl, Br, F, I, —CN, —NO₂, —OH,—CH₃, —CF₃, —NH₂, —C≡N, —C═N groups, —C—N—C— groups, —C—N—C(═O)— groups,—C—N—C(═O)—C—F, —C—N—C(═O)—C═C, substituted and unsubstituted alkylgroups, substituted and unsubstituted aryl groups, substituted andunsubstituted heterocyclyl groups, alkoxy groups, aryloxy groups,substituted and unsubstituted heterocyclylalkyl groups, substituted andunsubstituted aminoalkyl groups, substituted and unsubstitutedalkylaminoalkyl groups, substituted and unsubstituted dialkylaminoalkylgroups, substituted and unsubstituted arylaminoalkyl groups, substitutedand unsubstituted diarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted alkylaminogroups, substituted and unsubstituted arylamino groups, substituted andunsubstituted dialkylamino groups, substituted and unsubstituteddiarylamino groups, substituted and unsubstituted (alkyl)(aryl)aminogroups, —C(═O)H, —C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)O-alkylgroups, —C(═O)O-aryl groups, —C(═O)NH₂, —C(═O)NH(alkyl) groups,—C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂ groups, —C(═O)-aryl groups,—C(═O)NH₂, —C(═O)NH(alkyl) groups, —C(═O)NH(aryl) groups,—C(═O)N(alkyl)₂ groups, —C(═O)N(aryl)₂ groups, —C(═O)N(alkyl)(aryl)groups, —C(═O)O-alkyl groups, —C(═O)O-aryl groups, —C(═O)-heterocyclylgroups, —C(═O)—O-heterocyclyl groups, —C(═O)NH(heterocyclyl) groups,—C(═O)—N(heterocyclyl)₂ groups, —C(═O)—N(alkyl)(heterocyclyl) groups,—C(═O)—N(aryl)(heterocyclyl) groups, substituted and unsubstitutedheterocyclylaminoalkyl groups, substituted and unsubstitutedhydroxyalkyl groups, substituted and unsubstituted alkoxyalkyl groups,substituted and unsubstituted aryloxyalkyl groups, and substituted andunsubstituted heterocyclyloxyalkyl groups, substituted and unsubstituteddiheterocyclylaminoalkyl, substituted and unsubstituted(heterocyclyl)(alkyl)aminoalkyl, substituted and unsubstituted(heterocyclyl) (aryl)aminoalkyl, substituted and unsubstitutedalkoxyalkyl groups, substituted and unsubstituted hydroxyalkyl groups,substituted and unsubstituted aryloxyalkyl groups, and substituted andunsubstituted heterocyclyloxyalkyl groups; -(alkyl)(aryl)aminoalkylgroups, —C(═O)-heterocyclyl groups, —C(═O)—O-heterocyclyl groups,—C(═O)NH(heterocyclyl) groups, —C(═O)—N (heterocyclyl)₂ groups,—C(═O)—N(alkyl)(heterocyclyl) groups, —C(═O)—N(aryl)(heterocyclyl)groups, substituted and unsubstituted heterocyclylaminoalkyl groups,substituted and unsubstituted hydroxyalkyl groups, substituted andunsubstituted alkoxyalkyl groups, substituted and unsubstitutedaryloxyalkyl groups, and substituted and unsubstitutedheterocyclyloxyalkyl groups, —NH(alkyl) groups, —NH(aryl) groups,—N(alkyl)₂ groups, —N(aryl)₂ groups, —N(alkyl)(aryl) groups,—NH(heterocyclyl) groups, —N(heterocyclyl)(alkyl) groups,—N(heterocyclyl)(aryl) groups, —N(heterocyclyl)₂ groups, substituted andunsubstituted alkyl groups, substituted and unsubstituted aryl groups,substituted and unsubstituted alkoxy groups, substituted andunsubstituted aryloxy groups, substituted and unsubstituted heterocyclylgroups, —NHOH, —N(alkyl)OH groups, —N(aryl)OH groups, —N(alkyl)O-alkylgroups, —N(aryl)O-alkyl groups, —N(alkyl)O-aryl groups, and—N(aryl)O-aryl groups;

R⁸ is selected from the group consisting of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,H, absent, —C═C, substituted and unsubstituted heterocyclyl groups,substituted and unsubstituted aryl groups, substituted and unsubstitutedheterocyclyl(R⁹) groups, substituted and unsubstituted heterocyclyl(R¹⁰)groups, substituted and unsubstituted heterocyclyl(R¹¹) groups,substituted and unsubstituted heterocyclyl(R⁹)(R¹⁰) groups, substitutedand unsubstituted heterocyclyl(R⁹)(R¹¹) groups, substituted andunsubstituted heterocyclyl(R¹⁰)(R¹¹) groups, substituted andunsubstituted heterocyclyl(R⁹)(R¹⁰)(R¹¹) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R⁹) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R¹⁰) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R¹¹) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R⁹)(R¹⁰) groups, substituted andunsubstituted —C(═O)-heterocyclyl (R⁹)(R¹¹) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R¹⁰)(R¹¹) groups, substituted andunsubstituted —C(═O)-heterocyclyl(R⁹)(R¹⁰)(R¹¹) groups, substituted andunsubstituted aryl(R⁹) groups, substituted and unsubstituted aryl(R¹⁰)groups, substituted and unsubstituted aryl (R¹¹) groups, substituted andunsubstituted aryl (R⁹)(R¹⁰) groups, substituted and unsubstituted aryl(R⁹)(R¹¹) groups, substituted and unsubstituted aryl(R¹⁰)(R¹¹) groups,substituted and unsubstituted aryl (R⁹)(R¹⁰)(R¹¹) groups, substitutedand unsubstituted —C(═O)-aryl(R⁹) groups, substituted and unsubstituted—C(═O)-aryl(R¹⁰) groups, substituted and unsubstituted —C(═O)-aryl(R¹¹)groups, substituted and unsubstituted —C(═O)-aryl(R⁹)(R¹⁰) groups,substituted and unsubstituted aryl —C(═O)-aryl(R⁹)(R¹¹) groups,substituted and unsubstituted —C(═O)-aryl(R¹⁰)(R¹¹) groups, andsubstituted/unsubstituted —C(═O)-aryl(R⁹)(R¹⁰)(R¹¹) groups;

R⁹, R¹⁰, and R¹¹ may be the same or different and are independentlyselected from the group consisting of absent, H, Cl, Br, F, I, —CN,—NO₂, —OH, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R₁₂, C≡N, —C—N—C groups,—C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —C═N groups,substituted and unsubstituted alkyl groups, substituted andunsubstituted aryl groups, substituted and unsubstituted heterocyclylgroups, alkoxy groups, aryloxy groups, substituted and unsubstitutedheterocyclylalkyl groups, substituted and unsubstituted aminoalkylgroups, substituted and unsubstituted alkylaminoalkyl groups,substituted and unsubstituted dialkylaminoalkyl groups, substituted andunsubstituted arylaminoalkyl groups, substituted and unsubstituteddiarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted alkylaminogroups, substituted and unsubstituted arylamino groups, and substitutedand unsubstituted dialkylamino groups, substituted and unsubstitutedaminoalkyl groups, substituted and unsubstituted (alkyl)(aryl)aminoalkylgroups, substituted and unsubstituted alkylamino groups, substituted andunsubstituted arylamino groups, substituted and unsubstituteddialkylamino groups, substituted and unsubstituted diarylamino groups,substituted and unsubstituted (alkyl)(aryl)amino groups, —C(═O)H,—C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)O-alkyl groups,—C(═O)O-aryl groups, —C(═O)NH₂, —C(═O)NH(alkyl) groups, —C(═O)NH(aryl)groups, —C(═O)N(alkyl)₂ groups, —C(═O)-aryl groups, —C(═O)NH₂,—C(═O)NH(alkyl) groups, —C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂ groups,—C(═O)N(aryl)₂ groups, —C(═O)N(alkyl)(aryl) groups, —C(═O)O-alkylgroups, —C(═O)O-aryl groups, —C(═O)-heterocyclyl groups,—C(═O)—O-heterocyclyl groups, —C(═O)NH(heterocyclyl) groups,—C(═O)—N(heterocyclyl)₂ groups, —C(═O)—N(alkyl)(heterocyclyl) groups,—C(═O)—N (aryl)(heterocyclyl) groups, substituted and unsubstitutedheterocyclylaminoalkyl groups, substituted and unsubstituted cyanogroups, substituted and unsubstituted pyrimidinyl groups, substitutedand unsubstituted cyano(aryl) groups, substituted and unsubstitutedcyano (heterocyclyl) groups, and substituted and unsubstitutedcyano-pyrimidinyl groups;

R¹² is selected from the group consisting of absent, H, Cl, Br, F, I,—CN, —NO₂, —OH, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R¹², —C≡N, —C—N—Cgroups, —C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —C═N groups,—C(═O)— groups, —C(═O)—C— groups, —C(═O)—C═C, —S(═O)₂— groups,—S(═O)₂—C— groups, —S(═O)₂—C═C— groups, —S(═O)₂—C═C—CH₃, alkoxy groups,aryloxy groups, substituted and unsubstituted amidinyl groups,substituted and unsubstituted guanidinyl groups, substituted andunsubstituted primary, secondary, and tertiary alkyl groups, substitutedand unsubstituted aryl groups, substituted and unsubstituted alkenylgroups, substituted and unsubstituted alkynyl groups, substituted andunsubstituted heterocyclyl groups, substituted and unsubstitutedaminoalkyl groups, substituted and unsubstituted alkylaminoalkyl groups,substituted and unsubstituted dialkylaminoalkyl groups, substituted andunsubstituted arylaminoalkyl groups, substituted and unsubstituteddiarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstitutedheterocyclylalkyl groups, substituted and unsubstituted cyano groups,substituted and unsubstituted pyrimidinyl groups, substituted andunsubstituted cyano(aryl) groups, substituted and unsubstitutedcyano(heterocyclyl) groups, and substituted and unsubstitutedcyano-pyrimidinyl groups;

Q¹ is selected from the group consisting of a direct bond, H, C, Cl, Br,F, I, —CN, —NO₂, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R¹², —C≡N, —C—N—Cgroups, —C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —C═N groups,—C(═O)— groups, —C(═O)—C— groups, —C(═O)—C═C, —CF₃, —C≡N, —C—N—C—groups, —C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —OH, alkoxygroups, aryloxy groups, substituted and unsubstituted alkyl groups,substituted and unsubstituted aryl groups, substituted and unsubstitutedheterocyclyl groups, alkoxy groups, aryloxy groups, methoxy groups,dimethoxy groups, methoxy phenol, methoxy phenol groups, dimethoxyphenol, dimethoxy phenol groups, dimethoxy benzene, dimethoxy benzenegroups, methoxymethyl benzyl groups, substituted and unsubstitutedaralkyl groups, —NH², substituted and unsubstituted heterocyclylalkylgroups, substituted/unsubstituted aminoalkyl groups, substituted andunsubstituted alkylaminoalkyl groups, substituted and unsubstituteddialkylaminoalkyl groups, substituted and unsubstituted arylaminoalkylgroups, substituted and unsubstituted diarylaminoalkyl groups,substituted and unsubstituted (alkyl)(aryl)aminoalkyl groups,substituted and unsubstituted alkylamino groups, substituted andunsubstituted arylamino groups, and substituted and unsubstituteddialkylamino groups, substituted and unsubstituted cyano groups,substituted and unsubstituted pyrimidinyl groups, substituted andunsubstituted cyano(aryl) groups, substituted and unsubstitutedcyano(heterocyclyl) groups, and substituted and unsubstitutedcyano-pyrimidinyl groups;

Q² is selected from the groups consisting of absent, H, Q¹, Q¹(Q³), —OH,alkoxy groups, aryloxy groups; and

Q³ is selected from the group consisting of absent, a direct bond, H, C,Cl, Br, F, I, —CN—NO₂, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R¹², —C≡N, —C—N—Cgroups, —C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —C═N groups,—C(═O)— groups, —C(═O)—C— groups, —C(═O)—C═C, —CF₃, —C≡N, —C—N—C—groups, —C—N—C(═O)— groups, —C—N—C(═O)—C—F, —C—N—C(═O)—C═C, —OH, alkoxygroups, alkoxy groups, aryloxy groups, methoxy groups, dimethoxy groups,methoxy phenol, methoxy phenol groups, dimethoxy phenol, dimethoxyphenol groups, dimethoxy benzene, dimethoxy benzene groups, substitutedand unsubstituted alkyl groups, substituted and unsubstituted arylgroups, and substituted and unsubstituted heterocyclyl groups. Thecontents of the foregoing paragraph are hereinafter referred to as“OXR”.

In illustrative embodiments, the structure of R⁸ has the followingformula:

where X is independently selected from C, N, O, S, and —CN;

R⁹, R¹⁰, and R¹¹ may be the same or different and are independentlyselected from the group consisting of H, C, N, O, S, Cl, Br, F, I, —CN,—NO₂, —OH, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R¹², —C≡N, —C—N—C(═O)—C—F,—C—N—C(═O)—C═C, substituted and unsubstituted alkyl groups, substitutedand unsubstituted aryl groups, substituted and unsubstitutedheterocyclyl groups, —OH, alkoxy groups, aryloxy groups, substituted andunsubstituted heterocyclylalkyl groups, substituted and unsubstitutedaminoalkyl groups, substituted and unsubstituted alkylaminoalkyl groups,substituted and unsubstituted dialkylaminoalkyl groups, substituted andunsubstituted arylaminoalkyl groups, substituted and unsubstituteddiarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted alkylaminogroups, substituted and unsubstituted arylamino groups, and substitutedand unsubstituted dialkylamino groups, substituted and unsubstitutedcyano groups, substituted and unsubstituted pyrimidinyl groups,substituted and unsubstituted cyano(aryl) groups, substituted andunsubstituted cyano(heterocyclyl) groups, and substituted andunsubstituted cyano-pyrimidinyl groups; and

R¹² is selected from the group consisting of —C(═O)— groups, —C(═O)—C—groups, —C(═O)—C═C, —S(═O)₂— groups, —S(═O)₂—C— groups, —S(═O)₂—C═C—groups, —S(═O)₂—C═C—CH₃, —OH, alkoxy groups, aryloxy groups, substitutedand unsubstituted amidinyl groups, substituted and unsubstitutedguanidinyl groups, substituted and unsubstituted primary, secondary, andtertiary alkyl groups, substituted and unsubstituted aryl groups,substituted and unsubstituted alkenyl groups, substituted andunsubstituted alkynyl groups, substituted and unsubstituted heterocyclylgroups, substituted and unsubstituted aminoalkyl groups, substituted andunsubstituted alkylaminoalkyl groups, substituted and unsubstituteddialkylaminoalkyl groups, substituted and unsubstituted arylaminoalkylgroups, substituted and unsubstituted diarylaminoalkyl groups,substituted and unsubstituted (alkyl)(aryl)aminoalkyl groups,substituted and unsubstituted heterocyclylalkyl groups, substituted andunsubstituted cyano groups, substituted and unsubstituted pyrimidinylgroups, substituted and unsubstituted cyano(aryl) groups, substitutedand unsubstituted cyano(heterocyclyl) groups, and substituted andunsubstituted cyano-pyrimidinyl groups. The contents of the foregoingparagraph are hereinafter referred to as “QXR2”.

In illustrative embodiments, the structure of R⁸ is selected from GroupA as shown.

In illustrative embodiments, the structure of Q¹ and Q² is selected fromGroup B as shown below, —CH₃, —OH, —O—CH₃, —C—N—C(═O)—C═C, and—C—N—C(═O)—C—F.

In some embodiments, the compound of Structure 1 is a compound ofStructure 2, 2a, 3, 4 or 5, as shown below in Group C.

In illustrative embodiments, the compound of Structure 1, 2, 2a, 3, 4 or5 is administered orally, intravenously, subcutaneously, transdermally,intraperitoneally, or by inhalation. In illustrative embodiments, thekinase receptor is a receptor tyrosine kinase (RTK), and wherein the RTKis platelet derived growth factor receptor (PDGFR). In illustrativeembodiments, the PDGFR is platelet derived growth factor receptor-alpha(PDGFR-α) or platelet derived growth factor receptor-beta (PDGFR-β) orboth. In illustrative embodiments, the PDGFR is a homodimer orheterodimer selected from PDGFR-αα, PDGFR-ββ and PDGFR-αβ, or anycombination thereof. In illustrative embodiments, the inhibition of thePDGFR is effective in treating the pulmonary disorder, where thepulmonary disorder is pulmonary arterial hypertension (PAH), PAHassociated with plexiform and/or neointimal lesions, PAH associated withpulmonary fibrosis and/or progressive vaso-degeneration, abnormalfibroblast and/or myofibroblast proliferation, or pulmonary vasculardisorders associated with abnormal endothelial cell proliferation, orany combination thereof.

In illustrative embodiments, the inhibition is a combined inhibition ofboth the PDGFR-α and the PDGFR-β. In illustrative embodiments, theinhibition prevents activation of both the PDGFR-α and the PDGFR-β bymodulating cognate substrate interactions. In illustrative embodiments,the cognate substrate is selected from PDGFAA, PDGFBB and PDGFAB, or anycombination thereof. In illustrative embodiments, the pulmonary disorderis selected from pulmonary arterial hypertension (PAH), PAH associatedwith plexiform and/or neointimal lesions, PAH associated with pulmonaryfibrosis and/or progressive vasodegeneration, abnormal fibroblast and/ormyofibroblast proliferation, and pulmonary vascular disorders associatedwith abnormal endothelial cell proliferation.

In illustrative embodiments, the PAH selected from primary PAH,idiopathic PAH, heritable PAH, refractory PAH, BMPR2, ALK1, endoglinassociated with hereditary hemorrhagic telangiectasia, endoglin notassociated with hereditary hemorrhagic telangiectasia, drug-induced PAH,and toxin-induced PAH, PAH associated with systemic sclerosis, mixedconnective tissue disease, HIV, hepatitis, and/or portal hypertension.

In illustrative embodiments, the PAH is secondary to pulmonaryhypertension, congenital heart disease, hypoxia, chronic hemolyticanemia, newborn persistent pulmonary hypertension, pulmonaryveno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis(PCH), left heart disease pulmonary hypertension, systolic dysfunction,diastolic dysfunction, valvular disease, lung disease, interstitial lungdisease, pulmonary fibrosis, schistosomiasis, chronic obstructivepulmonary disease (COPD), sleep-disordered breathing, alveolarhypoventilation disorders, chronic exposure to high altitude,developmental abnormalities, chronic thromboembolic pulmonaryhypertension (CTEPH), pulmonary hypertension with unclear multifactorialmechanisms, hematologic disorders, myeloproliferative disorders,splenectomy, systemic disorders, sarcoidosis, pulmonary Langerhans cellhistiocytosis, lymphangioleimoyomatosis, neurofibromatosis, vasculitis,metabolic disorders, glycogen storage disease, Gaucher disease, thyroiddisorders, tumoral obstruction, fibrosing mediastinitis, and/or chronicrenal failure on dialysis.

In illustrative embodiments, the pulmonary disorder is associated withabnormal: right ventricular systolic pressure (RVSP); pulmonarypressure; cardiac output; right ventricular (RV) hypertrophy; and/orpulmonary arterial (PA) hypertrophy. In illustrative embodiments, thecompound of Structure 1 possesses an IC₅₀ of less than 300 nM for thekinase receptor. In illustrative embodiments, the kinase receptor isplatelet derived growth factor receptor-alpha (PDGFR-α) or plateletderived growth factor receptor-beta (PDGFR-β) or both, and where thepulmonary disorder is pulmonary arterial hypertension. In illustrativeembodiments, the inhibition occurs through a non-covalent interaction.In illustrative embodiments, the inhibition occurs through a covalentinteraction.

In one aspect, the present disclosure provides a method of treatingpulmonary arterial hypertension (PAH) in a subject, including:modulating the phosphorylation-state of one or more downstream targetsof platelet derived growth factor receptor-alpha (PDGFR-α) or plateletderived growth factor receptor-beta (PDGFR-β) or both, where thedownstream target is any substrate phosphorylated as a result of thePDGFR-α and/or the PDGFR-β activation, by administering to the subject acompound of Structure 1, a tautomer, enantiomer, isomer or stereoisomerof the compound, a pharmaceutically acceptable salt of the compound,tautomer, enantiomer, isomer or stereoisomer of the compound, or anymixtures thereof, where the downstream target is selected from the groupconsisting of AKT, PDGFR, STAT3, ERK1 and ERK2, or any other downstreamtarget of the PDGFR-α and/or the PDGFR-β, and where the compound ofStructure 1 has the following formula:

where X is independently selected from C, N, O, S or —CN;

R¹, R², and R³ may be the same or different and are independentlyselected from the group consisting of H, C, N, O, S, Cl, Br, F, I, —CN,—NO₂, —OH, —CH₃, —CF₃, —C—N—C— groups, —C—N—C(═O)— groups, —C(═O)R⁸groups, —N—C(═O)R⁸ groups, —C—N—C(═O)R⁸ groups, substituted andunsubstituted R⁸ groups, substituted and unsubstituted R⁸ groupssubstituted with one or more of R⁹, R¹⁰, and R¹¹, substituted andunsubstituted amidinyl groups, substituted and unsubstituted guanidinylgroups, substituted and unsubstituted primary, secondary, and tertiaryalkyl groups, substituted and unsubstituted aryl groups, substituted andunsubstituted alkenyl groups, substituted and unsubstituted alkynylgroups, substituted and unsubstituted heterocyclyl groups, substitutedand unsubstituted aminoalkyl groups, substituted and unsubstitutedalkylaminoalkyl groups, substituted and unsubstituted dialkylaminoalkylgroups, substituted and unsubstituted arylaminoalkyl groups, substitutedand unsubstituted diarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstitutedheterocyclylalkyl groups, substituted and unsubstituted cyano groups,substituted and unsubstituted pyrimidinyl groups, substituted andunsubstituted cyano(aryl) groups, substituted and unsubstitutedcyano(heterocyclyl) groups, and substituted and unsubstitutedcyano-pyrimidinyl groups;

R⁴, R⁵, R⁶, and R⁷, may be the same or different and are independentlyselected from the group consisting of H, Cl, Br, F, I, —CN, —NO₂, —OH,—CH₃, —CF₃, —NH₂, —C≡N, —C═N groups, —C—N—C— groups, —C—N—C(═O)— groups,—C—N—C(═O)—C—F, —C—N—C(═O)—C═C, substituted and unsubstituted alkylgroups, substituted and unsubstituted aryl groups, substituted andunsubstituted heterocyclyl groups, alkoxy groups, aryloxy groups,substituted and unsubstituted heterocyclylalkyl groups, substituted andunsubstituted aminoalkyl groups, substituted and unsubstitutedalkylaminoalkyl groups, substituted and unsubstituted dialkylaminoalkylgroups, substituted and unsubstituted arylaminoalkyl groups, substitutedand unsubstituted diarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted alkylaminogroups, substituted and unsubstituted arylamino groups, substituted andunsubstituted dialkylamino groups, substituted and unsubstituteddiarylamino groups, substituted and unsubstituted (alkyl)(aryl)aminogroups, —C(═O)H, —C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)O-alkylgroups, —C(═O)O-aryl groups, —C(═O)NH₂, —C(═O)NH(alkyl) groups,—C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂ groups, —C(═O)-aryl groups,—C(═O)NH₂, —C(═O)NH(alkyl) groups, —C(═O)NH(aryl) groups,—C(═O)N(alkyl)₂ groups, —C(═O)N(aryl)₂ groups, —C(═O)N(alkyl)(aryl)groups, —C(═O)O-alkyl groups, —C(═O)O-aryl groups, —C(═O)-heterocyclylgroups, —C(═O)—O-heterocyclyl groups, —C(═O)NH(heterocyclyl) groups,—C(═O)—N(heterocyclyl)₂ groups, —C(═O)—N(alkyl)(heterocyclyl) groups,—C(═O)—N(aryl) (heterocyclyl) groups, substituted and unsubstitutedheterocyclylaminoalkyl groups, substituted and unsubstitutedhydroxyalkyl groups, substituted and unsubstituted alkoxyalkyl groups,substituted and unsubstituted aryloxyalkyl groups, and substituted andunsubstituted heterocyclyloxyalkyl groups, substituted and unsubstituteddiheterocyclylaminoalkyl, substituted and unsubstituted(heterocyclyl)(alkyl)aminoalkyl, substituted and unsubstituted(heterocyclyl) (aryl)aminoalkyl, substituted and unsubstitutedalkoxyalkyl groups, substituted and unsubstituted hydroxyalkyl groups,substituted and unsubstituted aryloxyalkyl groups, and substituted andunsubstituted heterocyclyloxyalkyl groups; -(alkyl)(aryl)aminoalkylgroups, —C(═O)-heterocyclyl groups, —C(═O)—O-heterocyclyl groups,—C(═O)NH(heterocyclyl) groups, —C(═O)—N (heterocyclyl)₂ groups,—C(═O)—N(alkyl)(heterocyclyl) groups, —C(═O)—N(aryl) (heterocyclyl)groups, substituted and unsubstituted heterocyclylaminoalkyl groups,substituted and unsubstituted hydroxyalkyl groups, substituted andunsubstituted alkoxyalkyl groups, substituted and unsubstitutedaryloxyalkyl groups, and substituted and unsubstitutedheterocyclyloxyalkyl groups, —NH(alkyl) groups, —NH(aryl) groups,—N(alkyl)₂ groups, —N(aryl)₂ groups, —N(alkyl)(aryl) groups,—NH(heterocyclyl) groups, —N(heterocyclyl)(alkyl) groups,—N(heterocyclyl)(aryl) groups, —N(heterocyclyl)₂ groups, substituted andunsubstituted alkyl groups, substituted and unsubstituted aryl groups,substituted and unsubstituted alkoxy groups, substituted andunsubstituted aryloxy groups, substituted and unsubstituted heterocyclylgroups, —NHOH, —N(alkyl)OH groups, —N(aryl)OH groups, —N(alkyl)O-alkylgroups, —N(aryl)O-alkyl groups, —N(alkyl)O-aryl groups, and—N(aryl)O-aryl groups;

where the structure of R8 has the following formula:

and where X is independently selected from C, N, O, S, or —CN;

R⁹, R¹⁰, and R¹¹ may be the same or different and are independentlyselected from the group consisting of H, C, N, O, S, Cl, Br, F, I, —CN,—NO₂, —OH, —CH₃, —CF₃, —NH₂, —C(═O)—, —C—N—R¹², —C≡N, —C—N—C(═O)—C—F,—C—N—C(═O)—C═C, substituted and unsubstituted alkyl groups, substitutedand unsubstituted aryl groups, substituted and unsubstitutedheterocyclyl groups, —OH, alkoxy groups, aryloxy groups, substituted andunsubstituted heterocyclylalkyl groups, substituted and unsubstitutedaminoalkyl groups, substituted and unsubstituted alkylaminoalkyl groups,substituted and unsubstituted dialkylaminoalkyl groups, substituted andunsubstituted arylaminoalkyl groups, substituted and unsubstituteddiarylaminoalkyl groups, substituted and unsubstituted(alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted alkylaminogroups, substituted and unsubstituted arylamino groups, and substitutedand unsubstituted dialkylamino groups, substituted and unsubstitutedcyano groups, substituted and unsubstituted pyrimidinyl groups,substituted and unsubstituted cyano(aryl) groups, substituted andunsubstituted cyano(heterocyclyl) groups, and substituted andunsubstituted cyano-pyrimidinyl groups;

R¹² is selected from the group consisting of —C(═O)— groups, —C(═O)—C—groups, —C(═O)—C═C, —S(═O)₂— groups, —S(═O)₂—C— groups, —S(═O)₂—C═C—groups, —S(═O)₂—C═C—CH₃, —OH, alkoxy groups, aryloxy groups, substitutedand unsubstituted amidinyl groups, substituted and unsubstitutedguanidinyl groups, substituted and unsubstituted primary, secondary, andtertiary alkyl groups, substituted and unsubstituted aryl groups,substituted and unsubstituted alkenyl groups, substituted andunsubstituted alkynyl groups, substituted and unsubstituted heterocyclylgroups, substituted and unsubstituted aminoalkyl groups, substituted andunsubstituted alkylaminoalkyl groups, substituted and unsubstituteddialkylaminoalkyl groups, substituted and unsubstituted arylaminoalkylgroups, substituted and unsubstituted diarylaminoalkyl groups,substituted and unsubstituted (alkyl)(aryl)aminoalkyl groups,substituted and unsubstituted heterocyclylalkyl groups, substituted andunsubstituted cyano groups, substituted and unsubstituted pyrimidinylgroups, substituted and unsubstituted cyano(aryl) groups, substitutedand unsubstituted cyano(heterocyclyl) groups, and substituted andunsubstituted cyano-pyrimidinyl groups; and

where the structure of Q¹ or Q² is selected from the group consisting of—CH₃, —OH, —O—CH₃, —C—N—C(═O)—C═C, —C—N—C(═O)—C—F,

The entire contents of the foregoing paragraph are hereinafter referredto as “QXR3”.

In illustrative embodiments, the structure of R⁸ is selected from theGroup A structures noted above in the Summary. In illustrativeembodiments, the modulation is a decrease of phosphorylated STAT3 tototal STAT3, diphosphorylated ERK1 to total ERK1, diphosphorylated ERK2to total ERK2, monophosphorylated ERK1 to total ERK1, phosphorylatedPDGFR to total PDGFR, or phosphorylated AKT to total AKT, or anycombination thereof, in the subject compared to the PSR in the subjectbefore the administering. In illustrative embodiments, the compound ofStructure 1 interacts with AKT at residues Thr308 and/or Ser473, orwhere the compound of Structure 1 interacts with one or more of thePDGFR-α, PDGFR-β, PDGFR-αα, PDGFR-ββ, and/or the PDGFR-αβ amino acidsselected from LYS627, VAL607, GLU644, MET648, HIS816, LEU809, ASP836,CYS814, ILE834, CYS835, PHE937, LYS634, VAL614, GLU651, MET655, HIS824,LEU817, ASP844, CYS822, ILE842, VAL658, ILE647, HIS816, ARG836, LYS634,GLU651, ALA632, HIS824, MET655, ARG825, CYS843, THR874, ARG817, VAL815,LEU651, LEU809, ILE657, THR681, ILE654, ARG825, ASP826, LEU658, LEU825,PHE837, LEU658, HIS824, CYS814, ILE654, ASP844, ILE842, and/or CYS843,or any combination thereof.

In some embodiments, the compound of Structure 1 is a compound selectedfrom the Group C structures noted above in the Summary. In illustrativeembodiments, the inhibition occurs through a non-covalent interaction.In illustrative embodiments, the inhibition occurs through a covalentinteraction. In illustrative embodiments, compound of Structure 1, atautomer, enantiomer, isomer or stereoisomer of the compound, apharmaceutically acceptable salt of the compound, tautomer, enantiomer,isomer or stereoisomer of the compound, or any mixtures thereof, fortreating one or more diseases associated with hyperproliferation,neoplasia, hypoplasia, hyperplasia, dysplasia, metaplasia, prosoplasia,desmoplasia, angiogenesis, inflammation, immunological state,metabolism, pulmonary function, and cardiovascular function bynon-selectively inhibiting a receptor tyrosine kinase (RTK) selectedfrom AKT, c-Kit, and/or PDGFR, where Structure 1 is as follows:

where X, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ (and R⁸, R⁹, R¹⁰, R¹, and R¹², ascontained therein), Q¹ and Q² (and Q³ as contained therein) are selectedfrom “XRQ3”, as noted above.

In illustrative embodiments, the structure of R⁸ is selected from theGroup A structures noted above on the Summary. In some embodiments, thecompound is a structure selected from the Group C structures noted abovein the Summary.

In illustrative embodiments, the compound of Structure 1 is administeredorally, intravenously, subcutaneously, transdermally, intraperitoneally,or by inhalation. In illustrative embodiments, the disease is selectedfrom the group consisting of cancer, metastatic cancer, HIV, hepatitis,PAH, primary PAH, idiopathic PAH, heritable PAH, refractory PAH, BMPR2,ALK1, endoglin associated with hereditary hemorrhagic telangiectasia,endoglin not associated with hereditary hemorrhagic telangiectasia,drug-induced PAH, and toxin-induced PAH, PAH associated with systemicsclerosis, and mixed connective tissue disease, pulmonary hypertension,congenital heart disease, hypoxia, chronic hemolytic anemia, newbornpersistent pulmonary hypertension, pulmonary veno-occlusive disease(PVOD), pulmonary capillary hemangiomatosis (PCH), left heart diseasepulmonary hypertension, systolic dysfunction, diastolic dysfunction,valvular disease, lung disease, interstitial lung disease, pulmonaryfibrosis, schistosomiasis, COPD, sleep-disordered breathing, alveolarhypoventilation disorders, chronic exposure to high altitude,developmental abnormalities, chronic thromboembolic pulmonaryhypertension (CTEPH), pulmonary hypertension with unclear multifactorialmechanisms, hematologic disorders, myeloproliferative disorders,splenectomy, systemic disorders, sarcoidosis, pulmonary Langerhans cellhistiocytosis, lymphangioleimoyomatosis, neurofibromatosis, metabolicdisorders, glycogen storage disease, Gaucher disease, thyroid disorders,tumor obstruction, fibrosing mediastinitis, and chronic renal failure ondialysis.

In illustrative embodiments, the salt is a chloride, hydrochloride,sulfate, phosphate, mesylate, bismesylate, tosylate, lactate, tartrate,malate, bis-acetate, citrate, or bishydrochloride salt. In illustrativeembodiments, the inhibition occurs through a non-covalent interaction.In some embodiments, the inhibition occurs through a covalentinteraction. In some embodiments, compound of Structure 1 possesses anIC₅₀ of less than 300 nM for the kinase receptor. In illustrativeembodiments, the treatment methods result in one or more of improvedexercise capacity, improved functional class, less shortness of breath,decreased hospitalization, decreased need for lung transplantation,decreased need for atrial septostomy, and increased longevity or overallsurvival. In some embodiments, the improved exercise capacity is anincreased 6 minute walk distance. In suitable embodiments, improvedfunctional class is an improvement from class IV to class III, II or I,or an improvement from class III to class II or I, or an improvementform class II to class I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that the IC₅₀ of Imatinib against PDGFRα is 71 nM, whileFIG. 1B shows an IC₅₀ for PK10453 against PDGFRα of 35 nM. FIG. 1C,furthermore, shows that the IC₅₀ of Imatinib for PDGFRPβ is 607 nM,while FIG. 1D shows an IC₅₀ for PK10453 against PDGFRPβ of 10.1 nM.

FIG. 2A shows that PDGFAA stimulation of pAKT(S473) in HLFs was blockedby PK10453 (▪) and Imatinib (▴) with a comparable IC₅₀ between 0.3-0.6μM. FIG. 2B shows that PDGFAA stimulation of pAKT(T308) in HLFs wasblocked by PK10453 (▪) and Imatinib (▴) with a comparable IC₅₀ between0.3-0.6 μM. FIG. 2C shows that PDGFBB stimulation of pAKT(Ser473) wasblocked by PK10453 (▪) with an IC₅₀ of 0.13 μM compared to 1.8 μM forImatinib (▴). FIG. 2D shows that PDGFBB stimulation of pAKT(Thr308) wasblocked by PK10453 (▪) with an IC₅₀ of 0.43 μM compared to 3.25 μM forimatinib (▴). FIG. 2E shows examples of ICWs for PDGFAA and PDGFBBstimulated AKT phosphorylation, PK10453 vs. Imatinib. The signal at 800nm is color coded green and represents the phospho-protein specificsignal; the signal at 700 nm is color coded red and represents signalfrom total AKT. As shown, the 800 and 700 nm signals are superimposed (§p<0.01; * p<0.001).

FIG. 3 depicts fluorescence images of frozen rat lung sections (rightupper, middle, and lower lobes) after 2 min of PK10453 (Structure 2) andIR780 tracer inhalation. Image acquisition occurred at 800 nm (green),which is the λ of IR780 detection, while image acquisition at 700 nm(red) represents tissue autofluorescence. Digital ruler intervals areshow (1 cm).

FIG. 4A is a pharmacokinetic (PK) graph concerning IV administeredPK10453 and associated concentrations in the lungs and plasma as afunction of time. FIG. 4B is a PK graph concerning INH administeredPK10453 and associated levels in the lungs and plasma per time.

FIG. 5A is a graph showing the effect of PK10453 on RV systolic pressurein the MCT model, where C (n=3), V (n=2), D2 (n=6), D4 (n=6), and D8(n=5) respectively represent control, vehicle, 2 min exposure, 4 minexposure, and 8 min exposure times, for two weeks, three times daily.Asterisks (*) indicate p<0.001 and section symbols (§) indicate p<0.05.FIG. 5B is a graph showing the effect of PK10453 on RV hypertrophy inthe MCT model, where inhalation treatments were initiated three weeksafter administration of MCT. C, D2, D4, and D8 respectively representcontrols, 2, 4, and 8 min exposure times, for two weeks three timesdaily. The asterisks (*) indicate p<0.001. FIG. 5C is a graph showingthe effect of PK10453 on RV systolic pressure (RVSP) in the rat MCTmodel: comparison of PK10453 to imatinib; # p<0.01. FIG. 5D shows theLumen/Media ratio in the MCT model of PK10453, Imatinib and vehicle:Vehicle (V, n=4): 0.55±0.1; PK10453 (D8, n=12): 0.94±0.08; Imatinib (18,n=5): 0.99±0.07; § p<0.05, # p<0.01.

FIG. 6A is a graph showing pulmonary artery systolic pressure measuredover time in ambulatory subjects using the MCT+PN model system withPK10453. V (n=5) and D4 (n=6) respectively represent vehicle and 4 minexposure to PK10453 (Structure 2) three times daily. Asterisks (*)indicate p<0.001 and section symbols (§) indicate p<0.01. FIG. 6B is agraph showing pulmonary artery systolic pressure measured over time inambulatory subjects using the MCT+PN model system with Imatinib.V=vehicle; I=Imatinib (p=NS).

FIG. 7A shoes that RV systolic pressure: V (n=9) RVS, 75.7±7.1 mm Hg, D4group (n=10) RVS 40.4±2.7 mm Hg, D8 (n=8) RVS 43±3.0 mm Hg (p<0.001 Vvs. D4 and V vs. D8). FIG. 7B shows RV hypertrophy was decreased bytreatment with PK10453 (Structure 2); (RV+IVS)/LV ratio: V (n=11); D4(n=13); D8 (n=7); *p<0.001, § p<0.05. FIG. 7C shows the rat MCT+PNmodel, the lumen area/media area ratio was greater in the D8 (n=5)treated groups compared to PK10453 D4 (n=6) and vehicle (n=6); *p<0.0001D8 vs. V, D8 vs. D4. FIG. 7D shows occlusion analyses, which wereperformed on the same animal samples used for the lumen/media ratioanalysis. The occlusion analysis showed a significant decrease in Grade2 (>50% occlusive) lesions in the D8 group (#p<0.01).

FIG. 8A shows a microscope image of neointimal lesions. FIG. 8B shows animage of PK10453 treated subjects. FIG. 8C shows a phosphoPDGFRPβ(pPDGFRβ) stain, vehicle treated animal, while FIG. 8D shows a pPDGFRPβstain for PK10453 (D8) treated animals.

FIG. 9 is a graph showing lumen area:media area, which is increase in D4(n=6) and D8 (n=5) treated groups compared to vehicle (n=6) via MCT+PNmodel. Symbol (§) is p=0.032 (D4 vs. V), symbol (‡) is p=0.028 (D8 vs.D4), and asterisk (*) indicates p=0.00014 (D8 vs. V).

FIG. 10A shows that pSTAT3 localized to the nuclei of endothelial cellsand perivascular cells with vehicle treatment. FIG. 10B shows lungpSTAT3 nuclear signal from a subject treated with Structure 2.

FIG. 11A. shows Grade 0 lesions characterized by early intraluminalendothelial cell proliferation and the presence of vascular smoothmuscle cells in the media using αSMC stain.

FIG. 11B shows a Grade 1-2 lesion with extensive intraluminalmyofibroblast-like cells, some endothelial cells, and partial fibrosisof the medial layer using αSMC stain. FIG. 11C shows advanced Grade 2lesions characterized by extensive intraluminal myofibroblast-likecells, endothelial proliferation, and completely fibrotic replacement ofthe medial layer using αSMC stain. FIG. 11D shows Grade 0 lesionscharacterized by early intraluminal endothelial cell proliferation andthe presence of vascular smooth muscle cells in the media using atrichrome stain. FIG. 11E shows a Grade 1-2 lesion with extensiveintraluminal myofibroblast-like cells, some endothelial cells, andpartial fibrosis of the medial layer using a trichrome stain. FIG. 11Fshows advanced Grade 2 lesions characterized by extensive intraluminalmyofibroblast-like cells, endothelial proliferation, and completelfibrotic replacement of the medial layer using a trichrome stain. FIG.1G shows Grade 0 lesions characterized by early intraluminal endothelialcell proliferation and the presence of vascular smooth muscle cells inthe media using vWF stain. FIG. 11H shows a Grade 1-2 lesion withextensive intraluminal myofibroblast-like cells, some endothelial cells,and partial fibrosis of the medial layer using vWF stain. FIG. 11I showsadvanced Grade 2 lesions characterized by extensive intraluminalmyofibroblast-like cells, endothelial proliferation, and completelfibrotic replacement of the medial layer using vWF stain.

FIG. 12A shows 40×PDGFAA signaling in a pulmonary arteriole. FIG. 12Bshows 40× PDGFBB signaling in a pulmonary arteriole. FIG. 12C shows 40×total PDGFRα signaling in a pulmonary arteriole. FIG. 12D shows 40×total PDGFRPβ signaling in a pulmonary arteriole. FIG. 12E shows 40×phosphoPDGFRα (pPDGFRα) signaling in a pulmonary arteriole. FIG. 12Fshows 40× phosphoPDGFRPβ (pPDGFRβ) signaling in a pulmonary arteriole.

FIG. 13A shows 20× immunohistochemistry for pPDGFRα signal in the media.FIG. 13B shows 40× immunohistochemistry for pPDGFRα signal in the media.The arrow points to a smooth muscle cell positive for pPDGFRα. FIG. 13Cshows 20× imaging that, in contrast to above, there was very littlesignal in the media for pPDGFRPβ. FIG. 13D shows 40× imaging that, incontrast to above, there was very little signal in the media forpPDGFRβ. Signal for pPDGFRβ is noted in peri-vascular cells (upperleft—FIG. 13C and FIG. 13D), and endothelial cells.

FIG. 14A shows pAKT (Thr308) and total AKT, with vehicle treatment. FIG.14B shows pAKT (Thr308) and total AKT with PK10453 treatment. FIG. 14Cshows pAKT (Ser473) and total AKT, with vehicle treatment. FIG. 14Dshows pAKT(Ser473) and total AKT with PK10453 treatment. FIG. 14E showsthat the pAKT(Thr308)/AKT ratio in lung extracts was not significantlydifferent between the groups (V=vehicle; D4=4 minute exposure 3×/day for2 weeks, D8=8 minute exposure 3×/day for two weeks, p=NS). FIG. 14Frepresents the pAKT(Ser473)/AKT ratio in lung extracts for D8 group vs.vehicle (V, n=5; D4, n=4; D8, n=5) § p<0.05 D8 vs. V.

FIG. 15A is a graph of the vehicle treated subjects. FIG. 15B is a graphof the PK10453 (Structure 2) treated subjects. FIG. 15C shows a graph ofPK10453 treatment, which decreased pSTAT3/STAT3 in the lungs of subjectsusing the MCT+PN model (n=4), where V represents vehicle, D4 represents4 min exposure times three times daily, and D8 represent 8 min exposuretimes for two weeks three times daily; 3×/day for two weeks PK10453.Asterisks (*) p=0.009 and section symbols (§) indicate p=0.024.

FIG. 16A shows results from experiments using Nanopro™ immunoassaylumograms for pERK1/2 in vehicle treated subjects. FIG. 16B showsresults from experiments using Nanopro™ immunoassay lumograms forpERK1/2 in PK10453 treated subjects. FIG. 16C shows results fromexperiments using Nanopro™ immunoassay lumograms for total ERK1/2 invehicle treated subjects. FIG. 16D shows results from experiments usingNanopro™ immunoassay lumograms for total ERK1/2 in PK10453 treatedsubjects, where PK10453 decreased ppERK1/ERK1. FIG. 16E showsppERK1/ERK1 in subjects as indicated. FIG. 16F shows pERK2/ERK2 asindicated. FIG. 16G shows ppERK2/ERK2 as indicated in the lungs. FIG.16H shows pERK2/ERK2 as indicated in the lungs. The n=4 for each group,while V represents vehicle, D4 represents 4 min exposure times, threetimes daily, and D8 represents 8 min exposure times of PK10453(Structure 2) for two weeks three times daily. Asterisks (*) p<0.0005; §p=0.045.

FIG. 17A shows the effects of imatinib, PK10453 (Structure 2), andPK10571 (Structure 2a) on PDGFAA-stimulated phosphorylation of ERK1.FIG. 17B shows the effects of imatinib, PK10453 (Structure 2), andPK10571 (Structure 2a) on PDGFBB-stimulated phosphorylation of ERK1.FIG. 17C shows the effects of imatinib, PK10453 (Structure 2), andPK10571 (Structure 2a) on PDGFAA-stimulated phosphorylation of ERK2.FIG. 17D shows the effects of imatinib, PK10453 (Structure 2), andPK10571 (Structure 2a) on PDGFBB-stimulated phosphorylation of ERK2.

FIG. 18A shows the IC₅₀ concentrations of PK10467 (Structure 3), PK10571(Structure 2a), and imatinib for PDGFBB-stimulated AKT phosphorylationin fetal human lung fibroblasts. FIG. 18B shows the IC₅₀ concentrationsof PK10453 (Structure 2) and PK10571 (Structure 2a) forPDGFBB-stimulated AKT phosphorylation in fetal human lung fibroblasts.FIG. 18C shows the IC₅₀ concentrations of PK10468 (Structure 4), PK10569(Structure 5), and imatinib for PDGFBB-stimulated AKT phosphorylation infetal human lung fibroblasts. FIG. 18D shows the chemical structures ofStructure 2, Structure 2a, Structure 3, Structure 4, and Structure 5.

FIG. 19 is a graphic representation of subject body weight in vehicleadministered and PK10453 (Structure 2) treated subjects, where squaresindicate vehicle treated (n=10), triangles indicate PK10453 D4 group(n=10), and diamonds indicate PK10453 D8 group (n=6).

FIG. 20 is a graph representing PAC40 telemetry transmitter data fromtransmitters implanted in the abdominal aorta for monitoring systemicblood pressure for seven days in ambulatory MCT exposed subjects treatedwith vehicle (n=3) or PK10453 (n=3).

DETAILED DESCRIPTION

The present disclosure relates to, inter alia, a novel class ofcompounds which function as kinase inhibitors. Likewise, methods forusing such compounds in the prevention and treatment of diseaseconditions are disclosed herein. The present disclosure further relatesto pharmaceutical formulations of the compounds, which possessprophylactic and/or therapeutic indications for subjects in need ofkinase inhibitors, e.g., patients afflicted with vascular disease,proliferative disorders, cancers, and related diseases or conditions, asfurther detailed below. The definitions of certain terms as used in thisspecification are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “an amino acid”includes a combination of two or more nucleic acids, and the like.Moreover, as used herein, the following abbreviations have certainmeanings as detailed below.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the enumerated value.

As used herein, the following PK compounds and structure designationsare used interchangeably throughout the application: PK10453=Structure2; PK10571=Structure 2a; PK10467=Structure 3; PK10468=Structure 4; andPK10569=Structure 5.

As used herein, the “administration” of an agent or drug, e.g., one ormore kinase inhibitor compounds, to a subject or subjects includes anyroute of introducing or delivering to a subject a compound to performits intended function. Administration can be carried out by any suitableroute, including orally, intranasally, by inhalation, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),rectally, or topically. Administration includes self-administration andthe administration by another. It is also to be appreciated that thevarious modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial”, which includes total butalso less than total treatment or prevention, and where somebiologically or medically relevant result is achieved.

As used herein, the terms “comparable” or “corresponding” in the contextof comparing two or more samples, responses to treatment, or drugs,refer to the same type of sample, response, treatment, and drugrespectively used in the comparison. For example, the phosphorylationstate or level of AKT (pAKT) in a sample can be compared to thephosphorylation state or level in another sample. In some embodiments,comparable samples may be obtained from the same individual at differenttimes. In other embodiments, comparable samples may be obtained fromdifferent individuals, e.g., a patient and a healthy individual. Ingeneral, comparable samples are normalized by a common factor forcontrol purposes.

As used herein, the term “composition” refers to a product withspecified ingredients in the specified amounts, as well as any productwhich results, directly or indirectly, from combination of the specifiedingredients in the specified amounts.

As used herein, the terms “drug,” “compound,” “active agent,” “agent,”“actives,” “pharmaceutical composition,” “pharmaceutical formulation,”and “pharmacologically active agent” are used interchangeably and referto any chemical compound, complex or composition, charged or uncharged,that is suitable for administration and that has a beneficial biologicaleffect, suitably a therapeutic effect in the treatment of a disease orabnormal physiological condition, although the effect may also beprophylactic in nature. The terms also encompass pharmaceuticallyacceptable, pharmacologically active derivatives of those active agentsspecifically mentioned herein, including, but not limited to, salts,esters, amides, prodrugs, active metabolites, analogs, and the like.When the terms “active agent,” “pharmacologically active agent,” and“API” (active pharmaceutical ingredient) are used, then, or when aparticular active agent is specifically identified, it is to beunderstood that applicants intend to include the active agent per se aswell as pharmaceutically acceptable, pharmacologically active salts,esters, amides, prodrugs, metabolites, analogs, etc.

As used herein, the terms “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” of acomposition, is a quantity sufficient to achieve a desired therapeuticand/or prophylactic effect, e.g., an amount which results in theprevention of, or a decrease in, the symptoms associated with a diseasethat is being treated. The amount of a composition of the inventionadministered to the subject will depend on the type and severity of thedisease and on the characteristics of the individual, such as generalhealth, age, sex, body weight and tolerance to drugs. It will alsodepend on the degree, severity and type of disease. The skilled artisanwill be able to determine appropriate dosages depending on these andother factors. The compositions of the present invention can also beadministered in combination with one or more additional therapeuticcompounds.

As used herein, the terms “irreversible” or “irreversibly” whenreferring to a kinase inhibitor means an inhibitor of the activity of akinase, tyrosine kinase, and/or RTK, which is covalently, i.e.,permanently, bound or associated with such a kinase.

As used herein, the term “neoplastic disease” refers to cancers of anykind and origin and precursor stages thereof. Accordingly, the term“neoplastic disease” includes the subject matter identified by the terms“neoplasia”, “neoplasm”, “cancer”, “pre-cancer” or “tumor.” A neoplasticdisease is generally manifest by abnormal cell division resulting in anabnormal level of a particular cell population. Likewise, because themonoclonal expansion of endothelial cells may refer to a “neoplasm” ofthe pulmonary arteriolar endothelial cells, PAH is also encompassedwithin the foregoing terms. The abnormal cell division underlying aneoplastic disease, moreover, is typically inherent in the cells and nota normal physiological response to infection or inflammation. In someembodiments, neoplastic diseases for diagnosis using methods providedherein include carcinoma.

As used herein, the term “non-selective”, when referring to a kinaseinhibitor or receptor kinase inhibitor, means an inhibitor of theactivity of a kinase, tyrosine kinase, domain, and/or RTK, which is notsolely specific to a single kinase, receptor, tyrosine kinase, RTK ordomain, i.e., a cognate target, but within the context of inhibiting asingle kinase, receptor, tyrosine kinase, RTK, domain, etc., e.g., forPDGFR, the inhibitor is non-specific with respect to affinities and/orIC₅₀ concentrations for the kinase, receptor, tyrosine kinase, RTK,domain, etc. For example, PK10453 (Structure 2) targets PDGFR,non-selectively, by inhibiting both PDGFR-β and PDGFR-α, isoforms, butnevertheless may still possesses a lower IC₅₀ for a receptor isoform,e.g., PDGFR-β.

As used herein, the term “pharmaceutically acceptable salt” includes asalt with an inorganic base, organic base, inorganic acid, organic acid,or basic or acidic amino acid. As salts of inorganic bases, theinvention includes, for example, alkali metals such as sodium orpotassium; alkaline earth metals such as calcium and magnesium oraluminum; and ammonia. As salts of organic bases, the inventionincludes, for example, trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, and triethanolamine. As salts ofinorganic acids, the instant invention includes, for example,hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, andphosphoric acid. As salts of organic acids, the instant inventionincludes, for example, formic acid, acetic acid, trifluoroacetic acid,fumaric acid, oxalic acid, tartaric acid, maleic acid, lactic acid,citric acid, succinic acid, malic acid, methanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid. As salts of basicamino acids, the instant invention includes, for example, arginine,lysine and ornithine. Acidic amino acids include, for example, asparticacid and glutamic acid.

As used herein, the term “reference level” refers to a level of asubstance which may be of interest for comparative purposes. In someembodiments, a reference level may be a specified composition dosage asan average of the dose level from samples taken from a control subject.In other embodiments, the reference level may be the level in the samesubject at a different time, e.g., a time course of administering, suchas a level at 2, 4, 6, 8, and 10 minutes (min), etc.

As used herein, the terms “treating” or “treatment” or “alleviation”refer to both therapeutic treatment and prophylactic or preventativemeasures, wherein the objective is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for a disorder if, after receiving a therapeutic agentaccording to the methods of the present invention, the subject showsobservable and/or measurable reduction in or absence of one or moresigns and symptoms of a particular disease or condition.

As used herein, the term “unsubstituted alkyl” refers to alkyl groupsthat do not contain heteroatoms. Thus the phrase includes straight chainalkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrasealso includes branched chain isomers of straight chain alkyl groups,including but not limited to, the following which are provided by way ofexample: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃,—C(CH₂CH₃)₃, —CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂,—CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂,—CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includescyclic alkyl groups such as cycloalkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl andsuch rings substituted with straight and branched chain alkyl groups asdefined above. The phrase also includes polycyclic alkyl groups such as,but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl andsuch rings substituted with straight and branched chain alkyl groups asdefined above. Thus, the phrase unsubstituted alkyl groups includesprimary alkyl groups, secondary alkyl groups, and tertiary alkyl groups.Unsubstituted alkyl groups may be bonded to one or more carbon atom(s),oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parentcompound. Preferred unsubstituted alkyl groups include straight andbranched chain alkyl groups and cyclic alkyl groups having 1 to 20carbon atoms. More preferred such unsubstituted alkyl groups have from 1to 10 carbon atoms while even more preferred such groups have from 1 to5 carbon atoms. In some embodiments, unsubstituted alkyl groups includestraight and branched chain alkyl groups having from 1 to 3 carbon atomsand include methyl, ethyl, propyl, and —CH(CH₃)₂.

As used herein, the term “substituted alkyl” refers to an unsubstitutedalkyl group as defined above in which one or more bonds to a carbon(s)or hydrogen(s) are replaced by a bond to non-hydrogen and non-carbonatoms such as, but not limited to, a halogen atom in halides such as F,Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxygroups, aryloxy groups, and ester groups; a sulfur atom in groups suchas thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonylgroups, and sulfoxide groups; a nitrogen atom in groups such as amines,amides, alkylamines, dialkylamines, arylamines, alkylarylamines,diarylamines, N-oxides, imides, and enamines; a silicon atom in groupssuch as in trialkylsilyl groups, dialkylarylsilyl groups,alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatomsin various other groups. Substituted alkyl groups also include groups inwhich one or more bonds to a carbon(s) or hydrogen(s) atom is replacedby a bond to a heteroatom such as oxygen in carbonyl, carboxyl, andester groups; nitrogen in groups such as imines, oximes, hydrazones, andnitriles. In suitable embodiments, substituted alkyl groups include,among others, alkyl groups in which one or more bonds to a carbon orhydrogen atom is/are replaced by one or more bonds to fluorine atoms.One example of a substituted alkyl group is the trifluoromethyl groupand other alkyl groups that contain the trifluoromethyl group. Otheralkyl groups include those in which one or more bonds to a carbon orhydrogen atom is replaced by a bond to an oxygen atom such that thesubstituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, orheterocyclyloxy group. Still other alkyl groups include alkyl groupsthat have an amine, alkylamine, dialkylamine, arylamine,(alkyl)(aryl)amine, diarylamine, heterocyclylamine,(alkyl)(heterocyclyl)amine, (aryl)(heterocyclyl)amine, ordiheterocyclylamine group.

As used herein, the term “unsubstituted aryl” refers to aryl groups thatdo not contain heteroatoms. Thus the term includes, but is not limitedto, groups such as phenyl, biphenyl, anthracenyl, naphthenyl by way ofexample. Although the phrase “unsubstituted aryl” includes groupscontaining condensed rings such as naphthalene, it does not include arylgroups that have other groups such as alkyl or halo groups bonded to oneof the ring members, as aryl groups such as tolyl are considered hereinto be substituted aryl groups as described below. Unsubstituted arylgroups may be bonded to one or more carbon, oxygen, nitrogen, and/orsulfur atom(s).

As used herein, the term “substituted aryl group” has the same meaningwith respect to unsubstituted aryl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. However, a substitutedaryl group also includes aryl groups in which one of the aromaticcarbons is bonded to one of the non-carbon or non-hydrogen atomsdescribed above and also includes aryl groups in which one or morearomatic carbons of the aryl group is bonded to a substituted and/orunsubstituted alkyl, alkenyl, or alkynyl group as defined herein. Thisincludes bonding arrangements in which two carbon atoms of an aryl groupare bonded to two atoms of an alkyl, alkenyl, or alkynyl group to definea fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus,the term “substituted aryl” includes, but is not limited to, tolyl andhydroxyphenyl, among others.

As used herein, the term “unsubstituted alkenyl” refers to straight andbranched chain and cyclic groups such as those described with respect tounsubstituted alkyl groups as defined above, except that at least onedouble bond exists between two carbon atoms. Non-limiting examplesinclude vinyl, —CH═C(H)(CH₃), —CH═C(CH₃)₂, —C(CH₃)═C(H)₂,—C(CH₃)═C(H)(CH₃), —C(CH₂CH₃)═CH₂, cyclohexenyl, cyclopentenyl,cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

As used herein, the term “substituted alkenyl” has the same meaning withrespect to unsubstituted alkenyl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. A substituted alkenylgroup includes alkenyl groups in which a non-carbon or non-hydrogen atomis bonded to a carbon double bonded to another carbon and those in whichone of the non-carbon/non-hydrogen atoms is bonded to a carbon notinvolved in a carbon double bond.

As used herein, the term “unsubstituted alkynyl” refers to straight andbranched chain groups such as those described with respect tounsubstituted alkyl groups as defined above, except that at least onetriple bond exists between two carbon atoms. Examples include, but arenot limited to, —C≡C(H), —C≡C(CH₃), —C≡C(CH₂CH₃), —C(H₂)C≡C(H),—C(H)₂C≡C(CH₃), and —C(H)₂C≡C(CH₂CH₃), among others.

As used herein, the term “substituted alkynyl” has the same meaning withrespect to unsubstituted alkynyl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. A substituted alkynylgroup includes alkynyl groups in which a non-carbon or non-hydrogen atomis bonded to a carbon triple bonded to another carbon and those in whicha non-carbon or non-hydrogen atom is bonded to a carbon not involved ina carbon triple bond.

As used herein, the term “unsubstituted aralkyl” refers to unsubstitutedalkyl groups as defined above in which a hydrogen or carbon bond of theunsubstituted alkyl group is replaced with a bond to an aryl group asdefined above. For example, methyl (—CH₃) is an unsubstituted alkylgroup. If a hydrogen atom of the methyl group is replaced by a bond to aphenyl group, such as if the carbon of the methyl were bonded to acarbon of benzene, then the compound is an unsubstituted aralkyl group,i.e., a benzyl group. Thus, the term includes, but is not limited to,groups such as benzyl, diphenylmethyl, and1-phenylethyl(-CH(C₆H₅)(CH₃)), among others.

As used herein, the term “substituted aralkyl” has the same meaning withrespect to unsubstituted aralkyl groups that substituted aryl groups hadwith respect to unsubstituted aryl groups. However, a substitutedaralkyl group also includes groups in which a carbon or hydrogen bond ofthe alkyl part of the group is replaced by a bond to a non-carbon or anon-hydrogen atom. Non-limiting examples of substituted aralkyl groupsinclude —CH₂C(═O)(C₆H₅), and —CH₂(2-methylphenyl), among others.

As used herein, the term “unsubstituted heterocyclyl” refers to botharomatic and nonaromatic ring compounds including monocyclic, bicyclic,and polycyclic ring compounds such as, but not limited to, quinuclidyl,containing 3 or more ring members of which one or more is a heteroatomsuch as, but not limited to, N, O, and S. Examples of heterocyclylgroups include, but are not limited to: unsaturated 3 to 8 memberedrings containing 1 to 4 nitrogen atoms such as, but not limited topyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridinyl,dihydropyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl, e.g.,4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl etc.,tetrazolyl, e.g., 1H-tetrazolyl, 2H tetrazolyl, etc.); saturated 3 to 8membered rings containing 1 to 4 nitrogen atoms such as, but not limitedto, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensedunsaturated heterocyclic groups containing 1 to 4 nitrogen atoms suchas, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl;unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl,oxadiazolyl, e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,5-oxadiazolyl, etc.; saturated 3 to 8 membered rings containing 1 to2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to,morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl,benzoxadiazolyl, benzoxazinyl, e.g. 2H-1,4-benzoxazinyl, etc.);unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1to 3 nitrogen atoms such as, but not limited to, thiazolyl,isothiazolyl, thiadiazolyl, e.g., 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.;saturated 3 to 8 membered rings containing 1 to 2 sulfur atoms and 1 to3 nitrogen atoms such as, but not limited to, thiazolodinyl; saturatedand unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atomssuch as, but not limited to, thienyl, dihydrodithiinyl,dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; unsaturatedcondensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms such as, but not limited to, benzothiazolyl,benzothiadiazolyl, benzothiazinyl (e.g. 2H-1,4-benzothiazinyl, etc.),dihydrobenzothiazinyl, e.g., 2H-3,4-dihydrobenzothiazinyl, etc.,unsaturated 3 to 8 membered rings containing oxygen atoms such as, butnot limited to furyl; unsaturated condensed heterocyclic ringscontaining 1 to 2 oxygen atoms such as benzodioxolyl, e.g.,1,3-benzodioxoyl, etc.; unsaturated 3 to 8 membered rings containing anoxygen atom and 1 to 2 sulfur atoms such as, but not limited to,dihydrooxathiinyl; saturated 3 to 8 membered rings containing 1 to 2oxygen atoms and 1 to 2 sulfur atoms such as 1,4-oxathiane; unsaturatedcondensed rings containing 1 to 2 sulfur atoms such as benzothienyl,benzodithiinyl; and unsaturated condensed heterocyclic rings containingan oxygen atom and 1 to 2 oxygen atoms such as benzoxathiinyl.Heterocyclyl group also include those described above in which one ormore S atoms in the ring is double-bonded to one or two oxygen atoms(sulfoxides and sulfones). For example, heterocyclyl groups includetetrahydrothiophene oxide and tetrahydrothiophene 1,1-dioxide. Preferredheterocyclyl groups contain 5 or 6 ring members. More preferredheterocyclyl groups include morpholine, piperazine, piperidine,pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole,tetrazole, thiophene, thiomorpholine, thiomorpholine in which the S atomof the thiomorpholine is bonded to one or more O atoms, pyrrole,homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole,quinuclidine, thiazole, isoxazole, furan, and tetrahydrofuran.

As used herein, the term “substituted heterocyclyl” refers to anunsubstituted heterocyclyl group as defined above in which one or moreof the ring members are bonded to a non-hydrogen atom such as describedabove with respect to substituted alkyl groups and substituted arylgroups. Examples, include, but are not limited to,2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl,N-alkyl piperazinyl groups such as 1-methyl piperazinyl,piperazine-N-oxide, N-alkyl piperazine N-oxides, 2-phenoxy-thiophene,and 2-chloropyridinyl among others. In addition, substitutedheterocyclyl groups also include heterocyclyl groups in which the bondto the non-hydrogen atom is a bond to a carbon atom that is part of asubstituted and unsubstituted aryl, substituted and unsubstitutedaralkyl, or unsubstituted heterocyclyl group. Examples include but arenot limited to 1-benzylpiperidinyl, 3-phenythiomorpholinyl,3-(pyrrolidin-1-yl)-pyrrolidinyl, and 4-(piperidin-1-yl)-piperidinyl.Groups such as N-alkyl substituted piperazine groups such as N-methylpiperazine, substituted morpholine groups, and piperazine N-oxide groupssuch as piperazine N-oxide and N-alkyl piperazine N-oxides are examplesof some substituted heterocyclyl groups. Groups such as substitutedpiperazine groups such as N-alkyl substituted piperazine groups such asN-methyl piperazine and the like, substituted morpholine groups, andN-oxide groups are examples of some substituted heterocyclyl groups thatare suited for various “R” groups.

As used herein, the term “unsubstituted heterocyclylalkyl” refers tounsubstituted alkyl groups as defined above in which a hydrogen orcarbon bond of the unsubstituted alkyl group is replaced with a bond toa heterocyclyl group as defined above. For example, methyl (—CH₃) is anunsubstituted alkyl group. If a hydrogen atom of the methyl group isreplaced by a bond to a heterocyclyl group, such as if the carbon of themethyl were bonded to carbon 2 of pyridine (one of the carbons bonded tothe N of the pyridine) or carbons 3 or 4 of the pyridine, then thecompound is an unsubstituted heterocyclylalkyl group.

As used herein, the term “substituted heterocyclylalkyl” has the samemeaning with respect to unsubstituted heterocyclylalkyl groups thatsubstituted aralkyl groups had with respect to unsubstituted aralkylgroups. However, a substituted heterocyclylalkyl group also includesgroups in which a non-hydrogen atom is bonded to a heteroatom in theheterocyclyl group of the heterocyclylalkyl group such as, but notlimited to, a nitrogen atom in the piperidine ring of a piperidinylalkylgroup. In addition, a substituted heterocyclylalkyl group also includesgroups in which a carbon bond or a hydrogen bond of the alkyl part ofthe group is replaced by a bond to a substituted and unsubstituted arylor substituted and unsubstituted aralkyl group.

As used herein, the term “unsubstituted alkylaminoalkyl” refers to anunsubstituted alkyl group as defined above in which a carbon or hydrogenbond is replaced by a bond to a nitrogen atom that is bonded to ahydrogen atom and an unsubstituted alkyl group as defined above. Forexample, methyl (—CH₃) is an unsubstituted alkyl group. If a hydrogenatom of the methyl group is replaced by a bond to a nitrogen atom thatis bonded to a hydrogen atom and an ethyl group, then the resultingcompound is —CH₂—N(H)(CH₂CH₃) which is an unsubstituted alkylaminoalkylgroup.

As used herein, the term “substituted alkylaminoalkyl” refers to anunsubstituted alkylaminoalkyl group as defined above except where one ormore bonds to a carbon or hydrogen atom in one or both of the alkylgroups is replaced by a bond to a non-carbon or non-hydrogen atom asdescribed above with respect to substituted alkyl groups except that thebond to the nitrogen atom in all alkylaminoalkyl groups does not byitself qualify all alkylaminoalkyl groups as being substituted.

As used herein, the term “unsubstituted dialkylaminoalkyl” refers to anunsubstituted alkyl group as defined above in which a carbon bond orhydrogen bond is replaced by a bond to a nitrogen atom which is bondedto two other unsubstituted alkyl groups as defined above.

As used herein, the term “substituted dialkylaminoalkyl” refers to anunsubstituted dialkylaminoalkyl group as defined above in which one ormore bonds to a carbon or hydrogen atom in one or more of the alkylgroups is replaced by a bond to a non-carbon and non-hydrogen atom asdescribed with respect to substituted alkyl groups. The bond to thenitrogen atom in all dialkylaminoalkyl groups does not itself qualifyall dialkylaminoalkyl groups as substituted.

As used herein, the term “unsubstituted alkoxy” refers to a hydroxylgroup (—OH) in which the bond to the hydrogen atom is replaced by a bondto a carbon atom of an otherwise unsubstituted alkyl group as definedabove. As used herein, the term “substituted alkoxy” refers to ahydroxyl group (—OH) in which the bond to the hydrogen atom is replacedby a bond to a carbon atom of an otherwise substituted alkyl group asdefined above.

As used herein, the term “unsubstituted heterocyclyloxy” refers to ahydroxyl group (—OH) in which the bond to the hydrogen atom is replacedby a bond to a ring atom of an otherwise unsubstituted heterocyclylgroup as defined above. As used herein, the term “substitutedheterocyclyloxy” refers to a hydroxyl group (—OH) in which the bond tothe hydrogen atom is replaced by a bond to a ring atom of an otherwisesubstituted heterocyclyl group as defined above. As used herein, theterm “unsubstituted heterocyclyloxyalkyl” refers to an unsubstitutedalkyl group as defined above in which a carbon bond or hydrogen bond isreplaced by an oxygen bond, which is bonded to an unsubstitutedheterocyclyl group.

As used herein, the term “substituted heterocyclyloxyalkyl” refers to anunsubstituted heterocyclyloxyalkyl group as defined above in which abond to a carbon or hydrogen group of the alkyl group of theheterocyclyloxyalkyl group is bonded to a non-carbon and non-hydrogenatom as described above with respect to substituted alkyl groups or inwhich the heterocyclyl group of the heterocyclyloxyalkyl group is asubstituted heterocyclyl group as defined above.

As used herein, the term “unsubstituted heterocyclylalkoxy” refers to anunsubstituted alkyl group as defined above in which a carbon bond orhydrogen bond is replaced by a bond to an oxygen atom which is bonded tothe parent compound, and in which another carbon or hydrogen bond of theunsubstituted alkyl group is bonded to an unsubstituted heterocyclylgroup as defined above. As used herein, the term “substitutedheterocyclylalkoxy” refers to an unsubstituted heterocyclylalkoxy groupas defined above in which a bond to a carbon or hydrogen group of thealkyl group of the heterocyclylalkoxy group is bonded to a non-carbonand non-hydrogen atom as described above with respect to substitutedalkyl groups or in which the heterocyclyl group of theheterocyclylalkoxy group is a substituted heterocyclyl group as definedabove. Further, a substituted heterocyclylalkoxy group also includesgroups in which a carbon bond or a hydrogen bond to the alkyl moiety ofthe group may be substituted with one or more additional substituted andunsubstituted heterocycles.

As used herein, the term “unsubstituted arylaminoalkyl” refers to anunsubstituted alkyl group as defined above in which a carbon bond orhydrogen bond is replaced by a bond to a nitrogen atom which is bondedto at least one unsubstituted aryl group as defined above.

As used herein, the term “substituted arylaminoalkyl” refers to anunsubstituted arylaminoalkyl group as defined above except where eitherthe alkyl group of the arylaminoalkyl group is a substituted alkyl groupas defined above or the aryl group of the arylaminoalkyl group is asubstituted aryl group except that the bonds to the nitrogen atom in allarylaminoalkyl groups does not by itself qualify all arylaminoalkylgroups as being substituted. However, substituted arylaminoalkyl groupsdoes include groups in which the hydrogen bonded to the nitrogen atom ofthe group is replaced with a non-carbon and non-hydrogen atom.

As used herein, the term “unsubstituted heterocyclylaminoalkyl” refersto an unsubstituted alkyl group as defined above in which a carbon orhydrogen bond is replaced by a bond to a nitrogen atom which is bondedto at least one unsubstituted heterocyclyl group as defined above. Asused herein, the term “substituted heterocyclylaminoalkyl” refers tounsubstituted heterocyclylaminoalkyl groups as defined above in whichthe heterocyclyl group is a substituted heterocyclyl group as definedabove and/or the alkyl group is a substituted alkyl group as definedabove. The bonds to the nitrogen atom in all heterocyclylaminoalkylgroups does not by itself qualify all heterocyclylaminoalkyl groups asbeing substituted.

As used herein, the term “unsubstituted alkylaminoalkoxy” refers to anunsubstituted alkyl group as defined above in which a carbon or hydrogenbond is replaced by a bond to an oxygen atom which is bonded to theparent compound and in which another carbon or hydrogen bond of theunsubstituted alkyl group is bonded to a nitrogen atom which is bondedto a hydrogen atom and an unsubstituted alkyl group as defined above.

As used herein, the term “substituted alkylaminoalkoxy” refers tounsubstituted alkylaminoalkoxy groups as defined above in which a bondto a carbon or hydrogen atom of the alkyl group bonded to the oxygenatom which is bonded to the parent compound is replaced by one or morebonds to a non-carbon and non-hydrogen atoms as discussed above withrespect to substituted alkyl groups and/or if the hydrogen bonded to theamino group is bonded to a non-carbon and non-hydrogen atom and/or ifthe alkyl group bonded to the nitrogen of the amine is bonded to anon-carbon and non-hydrogen atom as described above with respect tosubstituted alkyl groups. The presence of the amine and alkoxyfunctionality in all alkylaminoalkoxy groups does not by itself qualifyall such groups as substituted alkylaminoalkoxy groups.

As used herein, the term “unsubstituted dialkylaminoalkoxy” refers to anunsubstituted alkyl group as defined above in which a carbon or hydrogenbond is replaced by a bond to an oxygen atom which is bonded to theparent compound and in which another carbon or hydrogen bond of theunsubstituted alkyl group is bonded to a nitrogen atom which is bondedto two other similar or different unsubstituted alkyl groups as definedabove.

As used herein, the term “substituted dialkylaminoalkoxy” refers to anunsubstituted dialkylaminoalkoxy group as defined above in which a bondto a carbon or hydrogen atom of the alkyl group bonded to the oxygenatom which is bonded to the parent compound is replaced by one or morebonds to a non-carbon and non-hydrogen atoms as discussed above withrespect to substituted alkyl groups and/or if one or more of the alkylgroups bonded to the nitrogen of the amine is bonded to a non-carbon andnon-hydrogen atom as described above with respect to substituted alkylgroups. The presence of the amine and alkoxy functionality in alldialkylaminoalkoxy groups does not by itself qualify all such groups assubstituted dialkylaminoalkoxy groups.

As used herein, the term “protected” with respect to hydroxyl groups,amine groups, and sulfhydryl groups refers to forms of thesefunctionalities which are protected from undesirable reaction with aprotecting group known to those skilled in the art such as those setforth in Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P.G. M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999), which canbe added or removed using the procedures set forth therein. Examples ofprotected hydroxyl groups include, but are not limited to, silyl etherssuch as those obtained by reaction of a hydroxyl group with a reagentsuch as, but not limited to, t-butyldimethyl-chlorosilane,trimethylchlorosilane, triisopropylchlorosilane, triethylchlorosilane;substituted methyl and ethyl ethers such as, e.g., methoxymethyl ether,methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethylether, allyl ether, benzyl ether; esters such as, but not limited to,benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate.Non-limiting examples of protected amine groups include amides such as,formamide, acetamide, trifluoroacetamide, and benzamide; imides, such asphthalimide, and dithiosuccinimide; and others. Non-limiting examples ofprotected sulfhydryl groups include thioethers such as S-benzylthioether, and S-4-picolyl thioether; substituted S-methyl derivativessuch as hemithio, dithio and aminothio acetals, among others.

Overview

Various compounds have been found useful in treating certain diseasessuch as, e.g., cancer. For example, Gleevec® (imatinib mesylate or“imatinib”) is a compound that has shown efficacy in treating chronicmyeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Otherexperimental drugs include sorafenib and PNU-166196 for the respectivetreatment of renal cell carcinoma and leukemia. Although significantadvances have been made in the development of pharmaceuticalcompositions for treating certain cancers; new compounds, compositions,methods of treatment, and model systems for developing drugs arerequired for preventing and/or treating cancer and other diseases, e.g.,pulmonary-vascular disease such as pulmonary arterial hypertension(PAH). In particular, platelet derived growth factor (PDGF) receptortyrosine kinases are an attractive therapeutic target for PAH. The PDGFsignaling pathway is activated in human idiopathic PAH (iPAH) and inanimal models of the disease. For example, PDGFA, PDGFB, PDGFRα andPDGFRPβ mRNA expression is increased in small pulmonary arteries frompatients with iPAH compared to control subjects, and Western blotanalysis shows a significant increase in protein expression of PDGFR3 inPAH lungs.

The migration of PASMCs is inhibited by imatinib, a PDGFRα inhibitor.Imatinib also decreases RVSP and improved survival in the rat MCT modelof PAH. In several case reports of patients with refractory PAH, afavorable response to imatinib has been observed. See Ghofrani et al.,“Imatinib in pulmonary arterial hypertension patients with inadequateresponse to established therapy.” Am J Respir Crit Care Med. Vol.182:1171-7 (2010). The IMPRES trial, which examined the effect ofimatinib in patients with severe PAH, showed an improvement in the sixminute walk distance and in cardiopulmonary hemodynamics. However,orally administered imatinib may be associated with systemic sideeffects including gastrointestinal distress and bone marrow suppression.See Paniagua et al., “Imatinib for the treatment of rheumatic diseases.”Nat Clin Pract Rheumatol; Vol 3:190-1 (2007). To improve the therapeuticwindow, i.e., increase efficacy and decrease systemic side-effects, thepresent inventors employed inhalation delivery of kinase inhibitors forPAH.

Imatinib, moreover, was developed using an in vivo murine MCT modelsystem, which is an imperfect system concerning preclinical drugcandidate efficacy assessment at least because it is unreliable withrespect to expressing certain human disease phenotypes, e.g., thedevelopment of neointimal and/or plexiform lesions associated with PAH.Cool et al., “Pathogenesis and evolution of plexiform lesions inpulmonary hypertension associated with scleroderma and humanimmunodeficiency virus infection.” Hum Pathol. 28:434-442 (1997).Therefore, examining the effects of kinase inhibitors in more aggressivemodels presenting human disease phenotypes is essential for moreaccurately reflecting the pathology of the human disease and,consequently, the development of the next generation of compounds andcompositions for effectively treating human disease.

The present inventors have employed such a model, while furthercomparing the present compounds and therapies to imatinib. As furtherdetailed below, the present inventors performed efficacy studies using amurine monocrotaline (MCT) plus pneumonectomy (PN) model system(MCT+PN). This model imparts neointimal and/or plexiform lesionscharacteristic of human disease, e.g., PAH. To this end, for example,the pathologic signature of PAH consists of concentric and plexiformlesions in small precapillary pulmonary arterioles. See Cool et al.(1997); and Tuder et al., “Plexiform lesion in severe pulmonaryhypertension: association with glomeruloid lesion.” Am J Pathol159:382-383 (2001). Concentric lesions arise from the proliferation ofneointimal cells, which occlude the vessel lumen. It has been reportedthat these concentric obstructive neointimal lesions are composed ofmyofibroblasts and/or endothelial cells. See, e.g., Yi et al., Am JRespir Crit Care Med 162:1577-86 (2000). In addition, perivascularinfiltrates, consisting of T cells, B cells, and macrophages, have beenfound in plexogenic PAH. See Sakagami, “In vivo, in vitro and ex vivomodels to assess pulmonary absorption and disposition of inhaledtherapeutics for systemic delivery.” Adv Drug Deliv Rev 58:1030-1060(2006). Plexiform lesions, moreover, are characterized by disorganizedvascular channels that stain for endothelial cell markers, and suchlesions in lung samples from patients with idiopathic and/or primary PAHconsist of a monoclonal expansion of endothelial cells. Lee et al.,“Monoclonal endothelial cell proliferation is present in primary but notsecondary pulmonary hypertension.” J Clin Invest 101:927-934 (1998). Assuch, PAH of this type is essentially a “cancer” of pulmonary arteriolarendothelial cells (see id.), at least because in the initial or earlystages of the disease, an acute apoptotic loss of normal endothelialcells may result in the emergence and clonal expansion of apoptosisresistant endothelial cells. Lee et al. (1998). The neoplastic processassociated with PAH provides for not only kinase inhibitor treatment ofPAH, but also the development of new compounds, compositions, andmethods, via MCT+PN model determinations, with superior efficacy,potency and a broader spectrum of inhibition compared to previouslygenerated kinase inhibitors using inferior model systems, which maypossess a narrow selectivity for RTK inhibition, for the treatment ofneoplastic disease. Drug-kinase homology modeling ensures that suchinhibitors, including, for example, non-selective and irreversiblyderivatives thereof, target vulnerable kinase domains for optimalefficacy, as further described below.

Compound Synthesis

In one aspect, the present disclosure provides for the synthesis ofStructure I compounds, which are readily synthesized using theprocedures described in the following sections and as disclosed in WO2008/058341, which is hereby incorporated by reference in its entiretyand for all purposes as if fully set forth herein. Compounds ofStructure I, moreover, are typically prepared from starting materials,such as, e.g., dihaloheterocycle. The first step is a nucleophilicaromatic substitution to generate a monoamino-monohalo intermediate. Thenucleophilic aromatic substitution is typically carried out by additionof a primary or secondary amine to the di-halogenated heterocycle in asolvent such as ethanol, isopropanol, tert-butanol, dioxane, THF, DMF,ethoxyethanol, toluene or xylene. The reaction typically occurs atelevated temperature in the presence of excess amine or anon-nucleophilic base such as triethylamine or diisopropylethylamine, oran inorganic base such as potassium carbonate or sodium carbonate.

Alternatively, the amino substituent may be introduced through atransition metal catalyzed amination reaction. Typical catalysts forsuch transformations include Pd(OAc)₂/P(t-Bu)₃, Pd₂(dba)₃/BINAP andPd(OAc)₂/BINAP. These reactions are typically carried out in solventssuch as toluene or dioxane, in the presence of bases such as caesiumcarbonate or sodium or potassium tert-butoxide at temperatures rangingfrom room temperature to reflux. See, e.g., Hartwig and Angew, Chem.Int. Ed 37, 2046 (1998). The amines employed in the first step of thesynthesis of these compounds are obtained commercially or are preparedusing methods well known to those skilled in the art.α-alkylbenzylamines, moreover, may be prepared through reduction ofoximes. Typical reductants include lithium aluminium hydride, hydrogengas in the presence of palladium on charcoal catalyst, Zn in thepresence of hydrochloric acid, sodium borohydride in the presence of aLewis acid such as TiCb, ZrCU, NiCl₂ and MoO₃, or sodium borohydridewith Amberlyst H1 5 ion exchange resin and LiCl. α-Alkylbenzylamines mayalso be prepared by reductive amination of the corresponding ketones. Aclassical method for such a transformation is the Leuckart-Wallachreaction, though catalytic conditions (HCO₂NH₄, [(CH₃)₅C₅RhCl₂]₂) orother procedures, e.g., NH₄OAc, Na(CN)BH₃) are also used.α-Alkylbenzylamines may also be prepared from the correspondingα-alkylbenzyl alcohols. Such methods include derivatisation of thehydroxyl as a mesylate or tosylate and displacement with a nitrogennucleophile, such as phthalimide or azide which is converted to theprimary amine using conventional synthetic methods; or, displacement ofthe hydroxyl with a suitable nitrogen nucleophile under Mitsunobu-likeconditions. α-Alkylbenzyl alcohols can be prepared by reduction of thecorresponding ketones with a reducing agent such as sodium borohydridein a solvent such as methanol. Alternatively, α-alkylbenzyl alcohols canbe obtained through addition of an alkyl metal species (such as aGrignard reagent) to a benzaldehyde derivative, which is typicallyperformed at room temperature or below in solvents such astetrahydrofuran. α-Alkyl benzylamines of high optical purity may beprepared from chiral α-alkyl benzyl alcohols using the methods outlinedabove. The chiral α-alkyl benzyl alcohols may be obtained through chiralreduction of the corresponding ketones.

The monoamino-monohalo intermediate formed from the dihaloheterocycleand the amine described above, may then be further functionalized. Forexample, where the amine substituent bears an additional functionalgroup, this functional group may be derivatized or functionalized usingmethods well-known to those skilled in the art. For example, a freeprimary amino group could be further functionalized to an amide,sulphonamide or urea functionality, or could be alkylated to generate asecondary or tertiary amine derivative. Preferable methods for theformation of an amide include coupling the amine with a carboxylic acidusing coupling reagents such as dicyclohexylcarbodiimide,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, diisopropylcarbodiimideor carbonyldiimidazole in solvents such as dichloromethane,tetrahydrofuran or 1,4-dioxane. Alternatively, the acid component may beactivated by conversion to an acid chloride (using thionyl chloride,oxalyl chloride, bis(trichloromethyl) carbonate or cyanuric chloride) orto mixed anhydride species (using, for example, t-butyl chloroformate orisopropyl chloroformate) or to active ester intermediates (such asN-hydroxysuccinimidyl, pentafluorophenyl or p-nitrophenyl esters) priorto amine reaction.

The monoamino-monochloro intermediate may then be reacted in a palladiummediated cross-coupling reaction with a suitably functionalized couplingpartner to replace the halogen atom with an alternative moiety. Typicalcoupling partners are organoboronic acids or esters. See, e.g., Miyauraand Suzuki, Chem Rev. 952457 (1995); Stille, Chem., Int. Ed. Engl 25,508 (1986); Kumada et al., Org. Synth. Coll. Vol. 6, 407 (1998); and:Negishi, J. Organomet. Chem. 653, 34 (2002) for Suzuki coupling,organostannanes, Stille coupling, Grignard reagents, Kumada coupling,organozinc species, and Negishi coupling, respectively. The Suzukicoupling is the preferred coupling method and is typically performed ina solvent such as DME, THF, DMF, ethanol, propanol, toluene, or1,4-dioxane in the presence of a base such as potassium carbonate,lithium hydroxide, caesium carbonate, sodium hydroxide, potassiumfluoride or potassium phosphate. The reaction may be carried out atelevated temperatures and the palladium catalyst employed may beselected from Pd(PPh₃)₄, Pd(OAc)₂, [PdCl₂(dppf)], Pd₂(dba)₃/P(t-Bu)₃.

The monoamino-monochloro intermediate may also be subjected to a secondnucleophilic aromatic substitution reaction using similar conditions tothose outlined above. Those skilled in the art will appreciate that theorder of the reactions described for the syntheses above may be changedin certain circumstances and that certain functionalities may need to bederivatized, i.e., protected, in certain instances for the reactionsdescribed above to proceed with reasonable yield and efficiency. Thetypes of protecting functionality are well-known to those skilled in theart. The products formed from the reaction sequences described above maybe further derivatized using techniques well known to those skilled inthe art. The leaving group may be any suitable known type such as thosedisclosed in March, “Advanced Organic Chemistry: Reactions, Mechanismsand Structure.” 4th Ed. pp 352-7, John Wiley & Sons, NY (1992). In someembodiments, the leaving group is a halogen, e.g., chlorine.

Kinases

Protein kinases are a family of enzymes that catalyze thephosphorylation of specific residues in proteins. Such enzymes aregenerally categorized into three groups, those which preferentiallyphosphorylate serine and/or threonine residues, those whichpreferentially phosphorylate tyrosine residues, and those whichphosphorylate both tyrosine and Ser/Thr residues. Protein kinases aretherefore key elements in signal transduction pathways responsible fortransducing extracellular signals, including the action of cytokines ontheir receptors, to the nuclei, triggering various biological events.The many roles of protein kinases in normal cell physiology include cellcycle control including proliferation, differentiation, metabolism,apoptosis, cell mobility, mitogenesis, transcription, translation andother signaling processes.

Platelet derived growth factor receptor kinase (PDGFR) is one type ofRTK. The sequence of PDGFR can be found in GenBank, accession numberNM-002609 (mRNA) and NP-002600 (protein) and has been described, atleast, in Matsui, et al., “Isolation of a novel receptor cDNAestablishes the existence of two PDGF receptor genes” Science243(4892):800-804 (1989); Claesson-Welsh, L. et al. “cDNA cloning andexpression of a human platelet-derived growth factor (PDGF) receptorspecific for B-chain-containing PDGF molecules” Mol. Cell. Biol.8(8):3476-3486 (1988); and Gronwald, et al. PNAS. 85(10):3435-3439(1988).

Moreover, PDGFR's cognate binding ligand, PDGF, is a strong mitogenicfactor for cells of mesenchymal origin such as fibroblasts, smoothmuscle cells, and glial cells. PDGF is a 32 kDa protein heterodimerusually composed of two polypeptide chains, A and B, linked by disulfidebonds. In addition to the PDGF AB heterodimer, two homodimeric forms ofPDGF exist (AA and BB). During blood clotting and platelet adhesion, thePDGF is released from granules at sites of injured blood vessels,suggesting that PDGF may have a role in the repair of blood vessels.PDGF may stimulate migration of arterial smooth muscle cells from themedial to the intimal layer of the artery where the muscle cells mayproliferate. The cellular proliferation induced by all isoforms of PDGFis mediated by ligand binding to the PDGF receptor. The PDGF receptorbelongs to the class III tyrosine kinase family and consists of tworeceptor subtypes, termed type A (or type alpha), and type B (or typebeta), as detailed above. Other members of the PDGF receptor familyinclude CSF-IR, cKIT and FLT3. The two PDGF receptor isoforms may bedistinguished by their markedly different ligand binding specificities.PDGFP3 receptor binds only B-chain (isoforms BB and AB), while PDGFUreceptor can bind all forms of PDGF (isoforms containing A and/or Bchain). With the importance of PDGF-related processes to proliferationof endothelial cells and vascular smooth muscle, there are a range ofpathogenic processes that PDGFRPβ kinase inhibitors are useful for,e.g., disease prevention and treatment.

PDGF expression has been shown in a number of different solid tumors,from glioblastomas to prostate carcinomas. In these various tumor types,the biological role of PDGF signaling can vary from autocrinestimulation of cancer cell growth to more subtle paracrine interactionsinvolving adjacent stroma and angiogenesis. Therefore, inhibiting thePDGFR kinase activity with small molecules may interfere with tumorgrowth, angiogenesis, diseases with neoplastic etiologies, immunologicaland inflammatory diseases, hyperproliferative diseases including cancerand diseases involving neo-angiogenesis, renal and kidney diseases, boneremodeling diseases, metabolic diseases, vascular diseases, andpulmonary vascular diseases such as, e.g., PAH. Other diseases mediatedby PDGF, and thus involving its cognate receptors, include, for example,restenosis, including coronary restenosis after angioplasty,atherectomy, or other invasive methods of plaque removal, and renal orperipheral artery restenosis after the same procedures; vascularproliferative phenomena and fibrosis associated with other forms ofacute injury such as pulmonary fibrosis associated with adultrespiratory distress syndrome, renal fibrosis associated with nephritis,coronary stenosis associated with Kawasake's disease and vascularnarrowings associated with other arteritides such as Takayasha'sdisease; prevention of narrowings in vein grafts; prevention ofnarrowings due to accelerated smooth muscle cell migration andproliferation in transplanted organs, and other fibrotic processes, suchas scleroderma and myofibrosis and inhibition of tumor cellproliferation.

c-Kit is another receptor tyrosine kinase belonging to PDGF Receptorfamily and is normally expressed in hematopoietic progenitor, mast andgerm cells. c-kit expression has been implicated in a number of cancersincluding mast cell leukemia, germ cell tumors, small-cell lungcarcinoma, GIST, acute myelogenous leukemia (AML), neuroblastoma,melanoma, ovarian carcinoma, breast carcinoma. Smolich et al., Blood,97(5) 1413-21.

Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) aremembers of the mitogen-activated protein (MAP) kinase super family thatcan mediate cell proliferation and apoptosis. The Ras-Raf-MEK-ERKsignaling cascade controlling cell proliferation has been well studiedbut the mechanisms involved in ERK1/2-mediated cell death are largelyunknown. ERK1/2 translocates to the nucleus, but can also remain in thecytosol. Cytosolic retention of ERK1/2 denies access to thetranscription factor substrates that are responsible for the mitogenicresponse. In addition, cytosolic ERK1/2, besides inhibiting survival andproliferative signals in the nucleus, potentiates the catalytic activityof some proapoptotic proteins such as DAP kinase in the cytoplasm.Studies that further define the function of cytosolic ERK1/2 and itscytosolic substrates that enhance cell death will be essential toharness this pathway for developing effective treatments for cancer andchronic inflammatory diseases.

STAT3 is a member of the STAT protein family, which typical function inresponse to cytokines and growth factors. STAT family members arephosphorylated by the receptor associated kinases, and then form homo-or heterodimers that translocate to the cell nucleus where they act astranscription activators. STAT3 is activated through phosphorylation inresponse to various cytokines and growth factors including IFNs, EGF,ILS, IL6, HGF, LIF and BMP2. This protein mediates the expression of avariety of genes in response to cell stimuli, and thus plays a key rolein many cellular processes such as cell growth and apoptosis. The smallGTPase Racl has been shown to bind and regulate the activity of thisprotein, while PIAS3 has been shown to inhibit STAT3.

AKT (also known as PKB) is involved in the regulation of metabolism,cell survival, motility, transcription and cell-cycle progression. AKTbelongs to the AGC subfamily of the protein kinase superfamily, whichconsists of more than 500 members in humans. The AKT subfamily comprisesthree mammalian isoforms, Akt1, Akt2, and Akt3, which are products ofdistinct genes and share a conserved structure that includes threefunctional domains: an N-terminal pleckstrin homology (PH) domain, acentral kinase domain, and a C-terminal regulatory domain containing thehydrophobic motif (HM) phosphorylation site [FxxF(S/T)Y].

Kinase Inhibitors

In one aspect, the present disclosure provides compounds and methods ofinhibiting a kinase, e.g., a tyrosine kinase, such as a RTK, in asubject and/or a method of treating a biological condition mediated by,or associated with, a kinase, e.g., a tyrosine kinase, such as a RTK, ina subject. In some embodiments, the kinase is Cdc2 kinase, AKT, c-Kit,c-ABL, ERK1/2, STAT3, p60src, VEGFR3, PDGFRα, PDGFRP3, FGFR3, PDGFR-αα,PDGFR-ββ, PDGFR-αβ, FLT-3, Fyn, Lck, Tie-2, GSK-3, Cdk2, Cdk4, MEK1,NEK-2, CHK2, CKlε, Raf, CHK1, Rsk2, FMS (CSF-IR), KDR, EphA2, EphA3,EphA8, FLT1, FLT4, HCK, PTKS, RET, SYK, DDR1, DDR2 and PAR-1. Likewise,the kinase is a tyrosine kinase, such as, e.g., Cdc2 kinase, c-Kit,c-ABL, p60src, VEGFR3, PDGFRα, PDGFRP3, FGFR3, PDGFR-αα, PDGFR-ββ,PDGFR-αβ, FLT-3, Fyn, Lck, and/or Tie-2, in some embodiments. Themethods include administering to the subject a compound of Structure I,a tautomer of the compound, a pharmaceutically acceptable salt of thecompound, a pharmaceutically acceptable salt of the tautomer, ormixtures thereof.

Previously, various indolyl substituted compounds are shown to inhibitone or more kinases, as disclosed in WO 01/29025, WO 01/62251, and WO01/62252. Likewise, various benzimidazolyl compounds have recently beendisclosed in WO 01/28993. Such compounds are reported to be capable ofinhibiting, modulating, and/or regulating signal transduction of bothreceptor-type and non-receptor tyrosine kinases. Some of the disclosedcompounds contain a quinolone fragment bonded to the indolyl orbenzimidazolyl group. The synthesis of 4-hydroxy quinolone and 4-hydroxyquinoline derivatives has also been reported. For example, Ukrainets etal. have disclosed the synthesis of3-(benzimidazol-2-yl)-4-hydroxy-2-oxo-1,2-dihydroquinoline. Ukrainets etal., Tet. Lett. 42, 7747-48 (1995) has also disclosed the synthesis,anticonvulsive and antithyroid activity of other 4-hydroxy quinolonesand thio analogs such as1H-2-oxo-3-(2-benzimidazolyl)-4-hydoxyquinoline. Ukrainets et al.,Khimiya Geterotsiklicheskikh Soedinii, 1, 105-108 (1993). Othercompounds, moreover, such as, for example,4-Amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-onehas been described as an orally bioavailable benzimidazole-quinolinonethat exhibits inhibition of receptor tyrosine kinases that drive bothendothelial and tumor cell proliferation. The inhibitory effect wasshown on nine tyrosine kinases, FGFR1, FGFR3, VEGFR1, VEGFR2, VEGFR3,PDGFRP3, c-Kit, p60src, and FLT-3, as disclosed in WO 2005/047244.However, this compound does not significantly inhibit EGFR familykinases or insulin receptor kinases at pharmaceutically acceptabledoses.

Moreover,4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide(imatinib), as disclosed in US 2006/0154936, inhibits PDGFRα and βkinases, Abl, DDR, and c-KIT, as described in US 2011/0190313. Paragraph[0117] of US 2011/0190313, however, indicates that although imatinibappeared safe and well tolerated over a 6 month period, the primaryefficacy parameter (6MWD) did not improve in patients randomized toimatinib compared with placebo, despite significant improvement insecondary endpoints. A continuing need therefore exists for compoundsthat inhibit, kinases, e.g., tyrosine kinases, such as RTKs, at leastbecause of previous limitations, resistant disease phenotypes, and theneed for more effective kinase, e.g., RTK, inhibition, as furtherdetailed below. See US 2008/0268460.

Furthermore, the small molecules that were reported in Frey et al.(1998) were shown to irreversibly inhibit epidermal growth factorreceptor (EGFR) by covalently interacting with the receptor, whilealkylating a cysteine residue in the ATP binding pocket of the molecule.Indeed, Leproult et al., “Cysteine Mapping in Conformationally DistinctKinase Nucleotide Binding Sites: Application to the Design of SelectiveCovalent Inhibitors.” J. Med. Chem. 54, 1347-1355 (2011), discloses thatone approach to designing irreversible inhibitors is to exploit thenucleophilicity of a cysteine thiol group present in the target proteinvia systematic analysis of cysteine residues present in the nucleotidebinding site of kinases. Such an approach can facilitate irreversibleinhibition even when taking into consideration the different kinaseconformations and therefore improve dosing and toxicity. See id.

The cysteine mapping in Leproult et al. (2011) demonstrate that kinasesare potential targets for selective covalent inhibitors. An example isshown of the kinase inhibitor imatinib to which a chloroacetamide groupis added in the para position of the benzene ring. Peptide inhibitoradduct formation was shown for both Kit and PDGFU receptors. Id.However, other compounds failed to show similar covalent adducts.Chloroacetamide is shown as an example of an electrophile which can forma covalent bond with a cysteine residue. The general term “warhead” isused to mean an electrophilic trap for forming a covalent bond betweenthe inhibitor and the targeted protein kinase. Chloroacetamide as anelectrophile may be too reactive to have clinical utility and may havetoxicity for this reason. Leproult et al. (2011) nevertheless suggestthat less then optimal positioning of the electrophile could explain whya covalent bond may not form with other less reactive warheads.

The present disclosure provides for, inter alio, distinct warheadpositioning on RTK receptor inhibitors. In some embodiments,electrophiles other than those described by Leproult et al. (2011), wereemployed for increased efficacy. See Barf et al. (2012) and Oballa etal., “A generally applicable method for assessing the electrophilicityand reactivity of diverse nitrile-containing compounds.” Bioorg Med ChemLett 17:998-1002 (2007) (describing nitrile-containing electrophiles).Furthermore, Diller et al., J Med Chem 46:4638-4647 (2003) reported ahomology model of the PDGFβ receptor based on VEGFR2 (55% homology).

Molecular docking was previously employed by the inventors with respectto one aspect of the present invention by using homology models of RTK,based on homologous structures, e.g., PDGFα and PDGFPβ receptor homologyto c-Kit is 59% and 63%, respectively. In some embodiments, theintroduction of various electrophiles in a variety of positions withrespect to a RTK inhibitor, e.g., PDGFR inhibitor, scaffold provided thebases for further biochemical analyses. To this end, the spatialorientation of the inhibitor warheads, relative to the target cysteineresidues, can be analyzed to calculate the free energy of binding andestimated K_(i). In some embodiments, compounds with the lowest freeenergy of binding and closest proximity of the warhead to a cysteineresidue impart irreversible non-selective RTK inhibitors.

Accordingly, the present disclosure provides compounds of Structure 1,the enantiomer, isomer or stereoisomer of the compound, apharmaceutically acceptable salt of the compound, tautomer, enantiomer,isomer or stereoisomer of the compound, or any mixtures thereof, whichcovalently interact with a receptor tyrosine kinase (RTK), such as, forexample, PDGFR or c-Kit or both. In some embodiments, the PDGFR isselected from the group consisting of PDGFR-α, PDGFR-β, PDGFR-αα,PDGFR-ββ, and PDGFR-αβ as demonstrated via homology modeling.

Pharmaceutical Compositions

In one aspect, the present disclosure provides pharmaceuticalcompositions which include at least one of the compounds of Structure 1and a pharmaceutically acceptable carrier. The compositions of thepresent invention may contain other therapeutic agents as describedbelow, and may be formulated, for example, by employing conventionalsolid or liquid vehicles or diluents, as well as pharmaceuticaladditives of a type appropriate to the mode of desired administration,for example, excipients, binders, preservatives, stabilizers, flavors,etc., according to techniques such as those well known in the art ofpharmaceutical formulation.

Pharmaceutical compositions are typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral (e.g., intravenous, intradermal,intraperitoneal or subcutaneous), oral, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. For convenience of thepatient or treating physician, the dosing formulation can be provided ina kit containing all necessary equipment for a treatment course.

The compounds of the present disclosure are administered by any suitablemeans, for example, orally, such as in the form of tablets, capsules,granules or powders; sublingually; buccally; parenterally, such as bysubcutaneous, intravenous, intramuscular, intra(trans)dermal, orintracisternal injection or infusion techniques, e.g., as sterileinjectable aqueous or non-aqueous solutions or suspensions, nasally suchas by inhalation spray or insufflation, topically, such as in the formof a cream or ointment ocularly in the form of a solution or suspension,vaginally in the form of pessaries, tampons or creams, or rectally suchas in the form of suppositories, in unit dosage formulations containingnontoxic, pharmaceutically acceptable vehicles or diluents. Thecompounds may, for example, be administered in a form suitable forimmediate release or extended release Immediate release or extendedrelease may be achieved by the use of suitable pharmaceuticalcompositions comprising the present compounds, or, for extended release,by the use of devices such as subcutaneous implants or osmotic pumps.

For administration to the respiratory tract, e.g., inhalation, includingintranasal administration, the active compound may be administered byany of the methods and formulations employed in the art foradministration to the respiratory tract. Thus, the active compound maybe administered in the form of, e.g., a solution, suspension, or as adry powder. The agents according to this aspect of the present inventionmay also be administered directly to the airways in the form of anaerosol. For use as aerosols, the compounds of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The materials of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

The propellant-driven inhalation aerosols which may be used according tothe invention may also contain other ingredients such as co-solvents,stabilizers, surfactants, antioxidants, lubricants and pH adjusters. Thepropellant-driven inhalation aerosols according to the invention whichmay be used according to the invention may be administered usinginhalers known in the art, e.g., metered dose inhalers. As anotheralternative, the agents of the present invention may be administered tothe airways in the form of a lung surfactant formulation. The lungsurfactant formulation can include exogenous lung surfactantformulations (e.g., Infasurf (Forest Laboratories), Survanta® (RossProducts), and Curosurtf (DEY, California, USA) or synthetic lungsurfactant formulations (e.g., Exosurtf (GlaxoWellcome Inc.) and ALEC).These surfactant formulations are administered via airway instillation(i.e., after intubation) or intratracheally.

As a further alternative, the agents of the present invention may beadministered to the airways in the form of an inhalable powder. Thepowder formulation may include physiologically acceptable excipientssuch as monosaccharides (e.g. glucose or arabinose), disaccharides (e.g.lactose, saccharose and maltose), oligo- and polysaccharides (e.g.dextrane), polyalcohols (e.g. sorbitol, mannitol, xylitol), salts (e.g.sodium chloride, calcium carbonate) or mixtures of these excipients withone another. Preferably, mono- or disaccharides are used, while the useof lactose or glucose is preferred, particularly, but not exclusively,in hydrate form.

Within the scope of the inhalable powders according to the invention theexcipients have a maximum average particle size of up to 250 am,preferably between 10 and 150 jam, most preferably between 15 and 80 am.It may sometimes seem appropriate to add finer excipient fractions withan average particle size of 1 to 9 am to the excipients mentioned above.These finer excipients are also selected from the group of possibleexcipients listed hereinbefore. Finally, in order to prepare theinhalable powders according to the invention, micronised formulations,preferably with an average particle size of 0.5 to 10 am is added to theexcipient mixture. Processes for producing the inhalable powdersaccording to the invention by grinding and micronizing and by finallymixing the ingredients together are known from the prior art.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the active compound is typicallyconfigured to have a small particle size, e.g., approximately 5 micronsor less, via micronisation techniques and the like. Sustained releaseformulations of the active compound are employed in some embodiments.The active compound, in some embodiments, is administered by oralinhalation as a free-flow powder via inhaler.

The pharmaceutical composition and method of the present disclosurefurther include additional therapeutically active compounds (secondagents), as noted herein and/or known in the art, which are typicallyemployed for treating one or more pathological conditions in concertwith the compositions comprising compounds of Structure 1 of the presentdisclosure. The combination of therapeutic agents acts synergisticallyto effect the treatment or prevention of the various diseases,disorders, and/or conditions described herein. Such second agents,include, but are not limited to, of prostanoids, endothelin antagonists,cytoplasmic kinase inhibitors, receptor kinase inhibitors, endothelinreceptor antagonists, e.g., ambrisentan, bosentan, and sitaxsentan, PDE5(PDE-V) inhibitors, e.g., sildenafil, tadalafil, and vardenafil, calciumchannel blockers, e.g., amlodipine, felodipine, varepamil, diltiazem,and menthol, prostacyclin, treprostinil, iloprost, beraprost, nitricoxide, oxygen, heparin, warfarin, diuretics, digoxin, cyclosporins,e.g., cyclosporin A, CTLA4-Ig, antibodies such as ICAM-3, anti-IL-2receptor (Anti-Tac), anti-CD45RB, anti-CD2, anti-CD3 (OKT-3), anti-CD4,anti-CD80, anti-CD86, agents blocking the interaction between CD40 andgp39, such as antibodies specific for CD40 and/or gp39, i.e., CD 154,fusion proteins constructed from CD40 and gp39 (CD40 1 g and CD8gp39),inhibitors, such as nuclear translocation inhibitors, of NF-kappa Bfunction, such as deoxyspergualin (DSG), cholesterol biosynthesisinhibitors such as HMG CoA reductase inhibitors (lovastatin andsimvastatin), non-steroidal anti-inflammatory drugs (NSAIDs) such asibuprofen, aspirin, acetaminophen, leflunomide, deoxyspergualin,cyclooxygenase inhibitors such as celecoxib, steroids such asprednisolone or dexamethasone, gold compounds, beta-agonists such assalbutamol, LABAs such as salmeterol, leukotriene antagonists such asmontelukast, antiproliferative agents such as methotrexate, FK506(tacrolimus, Prograf), mycophenolate mofetil, cytotoxic drugs such asazathioprine, VP-16, etoposide, fludarabine, doxorubin, adriamycin,amsacrine, camptothecin, cytarabine, gemcitabine, fluorodeoxyuridine,melphalan and cyclophosphamide, antimetabolites such as methotrexate,topoisomerase inhibitors such as camptothecin, DNA alkylators such ascisplatin, kinase inhibitors such as sorafenib, microtubule poisons suchas paclitaxel, TNF-α inhibitors such as tenidap, anti-TNF antibodies orsoluble TNF receptor, hydroxy urea and rapamycin (sirolimus or Rapamune)or derivatives thereof.

The compounds of the invention may also be prepared as salts which arepharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent disclosure at least to the extent that such salts are useful asintermediates in the preparation of pharmaceutically acceptable salts.Examples of pharmaceutically acceptable salts include, but are notlimited to, sulfates, phosphates, mesylates, bismesylates, tosylates,lactates, tartrates, malates, bis-acetates, citrates, bishydrochloridesalts, salts of pharmaceutically acceptable cations such as sodium,potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acidaddition salts of pharmaceutically acceptable inorganic acids such ashydrochloric, orthophosphoric, sulfuric, phosphoric, nitric, carbonic,boric, sulfamic and hydrobromic acids; or salts of pharmaceuticallyacceptable organic acids such as acetic, propionic, butyric, tartaric,maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic,benzoic, succinic, oxalic, phenylacetic, methanesulfonic,trihalomethanesulfonic, toluenesulfonic, benzenesulfonic, isethionic,salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic,oleic, lauric, pantothenic, tannic, ascorbic, valeric and orotic acids.Salts of amine groups may also comprise quaternary ammonium salts inwhich the amino nitrogen atom carries a suitable organic group such asan alkyl, alkenyl, alkynyl or aralkyl moiety. The salts may be formed byconventional means, such as by reacting the free base form of thecompound with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin. In some embodiments, the salt is a sulfate, phosphate,mesylate, bismesylate, tosylate, lactate, tartrate, malate, bis-acetate,citrate, or bishydrochloride salt.

In some embodiments, the compounds of the present disclosure areadministered in a therapeutically effective amount. Such anadministration imparts that a compound of Structure 1 will elicit aresponse associated with, e.g., cells, tissues, fluids, of a subjectbeing sought by the clinician. In the treatment or prevention ofconditions mediated by, or associated with, kinase inhibition, e.g., RTKinhibition, an appropriate dosage level is administered. In someembodiments, from about 0.01 to 500 mg/kg of subject body weight per dayis administered in single or multiple doses. In accord, dosage levelsare from about 0.1 to about 250 mg/kg per day in some embodiments, whilein other embodiments from about 0.5 to about 100 mg/kg per day isadministered to the subject. Suitable dosage levels include, forexample, from about 0.01 to 250 mg/kg per day, from about 0.05 to 100mg/kg per day, or from about 0.1 to 50 mg/kg per day. Within this range,in some embodiments, the dosage is from about 0.05 to 0.5, 0.5 to 5 or 5to 50 mg/kg per day. For oral administration, the compositions areprovided in the form of tablets containing 1.0 to 1000 mg of the activeingredient, including, but not limited to, 1, 5, 10, 15, 20, 25, 50, 75,100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 mg ofthe active ingredient. The dosage may be selected, for example, to anydose within any of these ranges, for therapeutic efficacy and/orsymptomatic adjustment of the dosage to the subject being treated. Insome embodiments, the compounds of the present disclosure areadministered by inhalation as described in, e.g., U.S. Pat. No.8,257,741, U.S. Pat. No. 8,263,128, WO 2010/132827, WO 2010/102066, WO2012/040502, WO 2012/031129, and/or WO 2010/102065, from 1 to 20, 1 to15, 1 to 10, 1 to 5, 1 to 4, or 1 to 3 times daily, or once or twice perday. In some embodiments, the compounds of the present disclosure areadministered from 1 to 5 times daily.

In some embodiments, the unit dose is sufficient to provide one or moreof: (a) a C_(max) of about 1 to 5000 ng/mL of the compound In asubject's plasma or a C_(max) of about 1 to 5000 ng/mL of the compoundIn the subject's blood when it is administered to the subject; and (b)about 1 to 5000 ng/mL of the compound in a subject's plasma 24 h afteradministration or about 1 to 5000 ng/mL of the compound in the subject'sblood 24 h after administration to the subject.

The therapeutically effective amount of a compound of Structure 1, thetautomer of the compound, enantiomer, isomer or stereoisomer of thecompound, a pharmaceutically acceptable salt of the compound, tautomer,enantiomer, isomer or stereoisomer of the compound, or any mixturesthereof, is not associated with adverse side effects, in someembodiments. Such adverse side effects include, but are not limited to,decreased lung function, increased or decreased systemic blood pressure,immunocompromised, bone marrow suppression, anemia, hypoxia, in thesubject compared to the subject prior to the administering.

Prevention and Treatment of Disease

In one aspect, the present disclosure provides a compound of Structure1, a tautomer of the compound, enantiomer, isomer or stereoisomer of thecompound, a pharmaceutically acceptable salt of the compound, tautomer,enantiomer, isomer or stereoisomer of the compound, or any mixturesthereof for treating one or more diseases, where Structure 1 isdescribed herein.

The present disclosure accordingly provides compounds, compositions, andmethods of inhibiting kinases, e.g., tyrosine kinases, and methods oftreating biological conditions mediated by, or associated with, suchkinases. For example, the present disclosure provides methods ofinhibiting one or more kinases, such as, e.g., cell division cycle 2kinase (Cdc2 kinase), c-Kit, c-ABL, p60src, AKT, VEGFR3, PDGFRα,PDGFRPβ, PDGFR-αα, PDGFR-ββ, PDGFR-αβ, FGFR3, FLT-3, FYN oncogene kinaserelated to SRC, FGR, YES (Fyn), lymphocyte-specific protein tyrosinekinase (Lck), tyrosine kinase with Ig and EGF homology domains (Tie-2),FMS (CSF-IR), KDR, EphA2, EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK,DDR1, DDR2, glycogen synthase kinase 3 (GSK-3), cyclin dependent kinase2 (Cdk2), cyclin dependent kinase 4 (Cdk4), MEK1, NEK-2, CHK2, CKlε,Raf, checkpoint kinase 1 (CHK1), ribosomal S6 kinase 2 (Rsk2), andPAR-1. In particular, compounds, compositions, and methods of inhibitingtyrosine kinases, such as, e.g., cell division cycle 2 kinase (Cdc2kinase), ERK1/2, STAT3, AKT, c-Kit, c-ABL, p60src, VEGFR3, PDGFRα,PDGFRPβ, PDGFR-αα, PDGFR-ββ, PDGFR-αβ, FGFR3, FLT-3, FYN oncogene kinaserelated to SRC, FGR, YES (Fyn), lymphocyte-specific protein tyrosinekinase (Lck), tyrosine kinase with Ig and EGF homology domains (Tie-2),FMS (CSF-IR), KDR, EphA2, EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK,DDR1, and DDR2. In some embodiments, the tyrosine kinase is a receptortyrosine kinase (RTK), such as, e.g., PDGFR, PDGFR-αα, PDGFR-ββ,PDGFR-αβ, or c-Kit, or combinations thereof, are provided.

The present disclosure also provides compounds, compositions, andmethods of treating biological conditions mediated by, or associatedwith, kinases, e.g., tyrosine kinases, including Cdc2 kinase, c-Kit,AKT, c-ABL, ERK1/2, STAT3, p60src, VEGFR3, PDGFRα, PDGFRPβ, FGFR3,PDGFR-αα, PDGFR-ββ, PDGFR-αβ, FLT-3, Fyn, Lck, Tie-2, GSK-3, Cdk2, Cdk4,MEK1, NEK-2, CHK2, CKlε, Raf, CHK1, Rsk2, FMS (CSF-IR), KDR, EphA2,EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK, DDR1, DDR2 and PAR-1. Inparticular, the present disclosure provides compounds, compositions, andmethods of treating biological conditions mediated by, or associatedwith, tyrosine kinases, including, but not limited to, Cdc2 kinase, AKT,c-Kit, c-ABL, p60src, VEGFR3, PDGFRα, PDGFRPβ, PDGFR-αα, PDGFR-ββ,PDGFR-αβ, FGFR3, FLT-3, Fyn, Lck, Tie-2, FMS (CSF-IR), KDR, EphA2,EphA3, EphA8, FLT1, FLT4, HCK, PTK5, RET, SYK, DDR1, and DDR2. In someembodiments, the disease or condition mediated by, or associated with,one or more kinases is mediated by a RTK, such as, e.g., PDGFR,PDGFR-αα, PDGFR-ββ, PDGFR-αβ, or c-Kit, or combinations thereof.

The disease or condition mediated by, or associated with, one or morekinases of the present disclosure, includes, but is not limited to, PAH,primary PAH, idiopathic PAH, heritable PAH, refractory PAH, BMPR2, ALK1,endoglin associated with hereditary hemorrhagic telangiectasia, endoglinnot associated with hereditary hemorrhagic telangiectasia, drug-inducedPAH, and toxin-induced PAH, PAH associated with or secondary to one ormore of systemic sclerosis, mixed connective tissue disease, cancer,refractory cancer, metastatic cancer, neoplasia, hypoplasia,hyperplasia, dysplasia, metaplasia, prosoplasia, desmoplasia, angiogenicdisease, pulmonary function disorders, cardiovascular functiondisorders, HIV infection, hepatitis, portal hypertension, pulmonaryhypertension, congenital heart disease, hypoxia, chronic hemolyticanemia, newborn persistent pulmonary hypertension, pulmonaryveno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis(PCH), left heart disease pulmonary hypertension, systolic dysfunction,diastolic dysfunction, valvular disease, lung disease, interstitial lungdisease, pulmonary fibrosis, schistosomiasis, chronic obstructivepulmonary disease (COPD), sleep-disordered breathing, alveolarhypoventilation disorders, chronic exposure to high altitude,developmental abnormalities, chronic thromboembolic pulmonaryhypertension (CTEPH), pulmonary hypertension with unclear multifactorialmechanisms, hematologic disorders, myeloproliferative disorders,splenectomy, systemic disorders, sarcoidosis, pulmonary Langerhans cellhistiocytosis, lymphangioleimoyomatosis, neurofibromatosis, vasculitis,metabolic disorders, glycogen storage disease, Gaucher disease, thyroiddisorders, tumoral obstruction, fibrosing mediastinitis, and chronicrenal failure on dialysis; and diseases such as pulmonary hypertension,congenital heart disease, hypoxia, chronic hemolytic anemia, newbornpersistent pulmonary hypertension, pulmonary veno-occlusive disease(PVOD), pulmonary capillary hemangiomatosis (PCH), left heart diseasepulmonary hypertension, systolic dysfunction, diastolic dysfunction,valvular disease, lung disease, interstitial lung disease, pulmonaryfibrosis, schistosomiasis, chronic obstructive pulmonary disease (COPD),sleep-disordered breathing, alveolar hypoventilation disorders, chronicexposure to high altitude, developmental abnormalities, chronicthromboembolic pulmonary hypertension (CTEPH), pulmonary hypertensionwith unclear multifactorial mechanisms, hematologic disorders,myeloproliferative disorders, splenectomy, systemic disorders,sarcoidosis, pulmonary Langerhans cell histiocytosis,lymphangioleimoyomatosis, neurofibromatosis, vasculitis, metabolicdisorders, glycogen storage disease, Gaucher disease, thyroid disorders,tumoral obstruction, fibrosing mediastinitis, immunological andinflammatory diseases, hyperproliferative diseases, renal and kidneydiseases, bone remodeling diseases, metabolic diseases, vasculardiseases, and chronic renal failure on dialysis.

In one aspect, the present disclosure provides a method of treatingpulmonary arterial hypertension (PAH) in a subject or a biologicalcondition associated with PAH in a subject by administering to thesubject a therapeutically effective amount of a compound of Structure 1,a tautomer of the compound, a pharmaceutically acceptable salt of thecompound, a pharmaceutically acceptable salt of the tautomer, or amixture thereof, wherein a compound of Structure 1 is described herein.In some embodiments, the disease or condition mediated by, or associatedwith, one or more kinases of the present disclosure is selected form thegroup consisting of PAH, primary PAH, idiopathic PAH, heritable PAH,refractory PAH, drug-induced PAH, toxin-induced PAH, and PAH withsecondary diseases.

Pulmonary arterial hypertension (PAH) is a life-threatening diseasecharacterized by a marked and sustained elevation of pulmonary arterypressure. The disease results in right ventricular (RV) failure anddeath. Current therapeutic approaches for the treatment of chronicpulmonary arterial hypertension mainly provide symptomatic relief, aswell as some improvement of prognosis. Although postulated for alltreatments, evidence for direct anti-proliferative effects of mostapproaches is missing. In addition, the use of most of the currentlyapplied agents is hampered by either undesired side effects orinconvenient drug administration routes. Pathological changes ofhypertensive pulmonary arteries include endothelial injury,proliferation and hyper-contraction of vascular smooth muscle cells(SMCs), and fibroblast proliferation. PAH patient status, moreover, canbe assessed in accordance with the World Health Organization (WHO)classification (modified after the NY Association FunctionalClassification) as known in the art.

In some embodiments, the compounds of Structure 1 treat or prevent PAHin patients who failed prior therapy, especially after receiving atleast one prostanoid, endothelin antagonist or PDE V inhibitor. In otherembodiments, the compounds treat or prevent PAH in patients who are moreseverely affected, in particular in patients with Class II to Class IVfunctional status, or more severely Class III or IV functional status.In further embodiments, the compounds treat or prevent PAH in patientswho are harboring BMPR2 mutations.

The present disclosure provides methods of preventing or treatingsubjects afflicted with idiopathic or primary pulmonary hypertension,familial hypertension, pulmonary hypertension secondary to, but notlimited to, connective tissue disease, congenital heart defects(shunts), pulmonary fibrosis, portal hypertension, HIV infection, sicklecell disease, drugs and toxins, e.g., anorexigens, cocaine, chronichypoxia, chronic pulmonary obstructive disease, sleep apnea, andschistosomiasis, pulmonary hypertension associated with significantvenous or capillary involvement (pulmonary veno-occlusive disease,pulmonary capillary hemangiomatosis), secondary pulmonary hypertensionthat is out of proportion to the degree of left ventricular dysfunction,and/or persistent pulmonary hypertension in newborn babies, especiallyin subjects that previously failed prior PAH therapy.

In one aspect, the present disclosure provides a compound of Structure1, a tautomer of the compound, enantiomer, isomer or stereoisomer of thecompound, a pharmaceutically acceptable salt of the compound, tautomer,enantiomer, isomer or stereoisomer of the compound, or any mixturesthereof for treating one or more diseases associated withhyperproliferation, neoplasia, hypoplasia, hyperplasia, dysplasia,metaplasia, prosoplasia, desmoplasia, angiogenesis, inflammation,pulmonary function, and cardiovascular function, where a compound ofStructure 1 is described herein.

Hyperproliferative, immunological and inflammatory, metabolic, andvascular diseases, are known in the art, and such diseases, as describedin U.S. Provisional Patent No. 61/751,217, which is hereby incorporatedby reference in its entirety, are therapeutic targets for the compoundsand agents described herein.

Another aspect of the present disclosure related to a method ofpreventing or reducing elevated pulmonary pressure in a subject, byadministering to the subject a therapeutically effective amount of acompound of Structure 1, a tautomer of the compound, a pharmaceuticallyacceptable salt of the compound, a pharmaceutically acceptable salt ofthe tautomer, or a mixture thereof, where a compound of Structure 1 isdescribed herein. See, e.g., Summary. In some embodiments, the compoundsof Structure 1 treat or prevent a biological condition associated withPAH, such as, e.g., abnormal: right ventricular systolic pressure(RVSP); pulmonary pressure; cardiac output; right ventricular (RV)hypertrophy; and PA hypertrophy.

In some embodiments, the compounds of Structure 1 reduce pulmonarypressure associated with an increase in one or more of right ventricular(RV) function, pulmonary artery (PA) systolic pressure, and/or cardiacoutput in the subject compared to the subject prior to theadministering. In some embodiments, the reduction in pulmonary pressureis associated with a decrease in one or more of RV hypertrophy, PAhypertrophy, RVSP, sustained PA pressure, and the risk of stroke in thesubject compared to the subject prior to the administering. In someembodiments, the decrease is at least a 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% decrease. In someembodiments, the decrease is at least a 40% decrease.

A reduction in pulmonary pressure, in some embodiments, is notassociated with decreased lung function and/or increased systemic bloodpressure in the subject compared to the subject prior to theadministering. Methods for measuring lung function and blood pressureare known in the art. In one aspect, the present disclosure provides amethod of treating pulmonary arterial hypertension (PAH) in a subject,comprising: modulating the phosphorylation-state (“PS”) of one or moredownstream targets of platelet derived growth factor receptor-alpha orplatelet derived growth factor receptor-beta or both, wherein thedownstream target is any substrate phosphorylated as a result of thePDGFR-α and/or the PDGFR-β activation, by administering to the subject acompound of Structure 1, a tautomer, enantiomer, isomer or stereoisomerof the compound, a pharmaceutically acceptable salt of the compound,tautomer, enantiomer, isomer or stereoisomer of the compound, or anymixtures thereof, wherein the downstream target is selected from thegroup consisting of AKT, PDGFR, STAT3, ERK1 and ERK2, or any otherdownstream target of the PDGFR-u and/or the PDGFR-β, and wherein thecompound of Structure 1 is described herein. Phosphorylation stateprofiles for proteins, kinases/receptors, can be ascertain usingtechniques known in the art, such as, for example, Z-lyte kinase assays,Invitrogen Select Screen®, and other kinases assay's know in the art.

In suitable embodiments, the modulation of the kinase receptor activityis an inhibition of the kinase receptor activity. PDGFR, i.e., PDGFR-α,PDGFR-β, PDGFR-αα, PDGFR-ββ, and PDGFR-αβ, and/or c-Kit are examples ofRTKs that are inhibited in some embodiments of the present invention. Insome embodiments, the inhibition is at least a 0.001, 0.01, 0.1, 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or95% inhibition. In some embodiments, the PSR modulation is a modulationof one or more of AKT, STAT3, ERK1, ERK2, PDGF, and PDGFR i.e.,PDGFR-αα, PDGFR-ββ, and PDGFR-αβ. In some embodiments, the modulation ofPS is a decrease of phosphorylated STAT3 to total STAT3 in the subjectcompared to the PS in the subject prior to the administering. In someembodiments, the decrease is at least a 0.001, 0.01, 0.1, 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%decrease. In some embodiments, the modulation of PS is a decrease ofdiphosphorylated ERK1 to total ERK1 in the subject compared to the PS inthe subject prior to the administering. In some embodiments, thedecrease is at least a 0.001, 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% decrease. In otherembodiments, the modulation of PS is a decrease of diphosphorylated ERK2to total ERK2 in the subject compared to the PS in the subject prior tothe administering. In some embodiments, the decrease is at least a0.001, 0.01, 0.1, 1, 10, 50, 60, 70, 80, 85, 90, or 95% decrease.

In some embodiments, the modulation of PS is a decrease ofmonophosphorylated ERK1 to total ERK1 in the subject compared to the PSin the subject prior to the administering. In some embodiments, thedecrease is at least a 0.001, 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% decrease. In someembodiments, the modulation of PS is a decrease of phosphorylated PDGFRto total PDGFR in the subject compared to the PS in the subject prior tothe administering. In some embodiments, the decrease is at least a0.001, 0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, or 95% decrease. In some embodiments, the modulationof PS is a decrease of phosphorylated AKT to total AKT in the subjectcompared to the PS in the subject prior to the administering. In someembodiments, the decrease is at least a 0.001, 0.01, 0.1, 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%decrease.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way. The following is adescription of the materials and methods used throughout the examples,which illustrates that RTK signaling pathways are activated in humandisease conditions, e.g., PAH and in animal models of the disease.

Materials.

PK10453,(S)-N-(3-(1-((6-(4-hydroxy-3-methoxyphenyl)pyrazin-2yl)amino)ethyl)phenyl)-5-methylnicotinamide,i.e., Structure 2, was synthesized by Organix, Inc. (Woburn, Mass.).Human PA smooth muscle cells and cell culture media were obtained fromCell Applications, Inc. PDGFBB, para-toluene sulfonic acid, ammoniumhydroxide, and IR780 were obtained from Sigma Aldrich (St. Louis, Mo.).Imatinib mesylate was obtained from LC Laboratories (Woburn, Mass.).Human fetal lung fibroblasts (HLFs) were obtained from CellApplications, Inc., San Diego. DMEM medium was obtained from Mediatech(Manassas, Va.). PDGFAA, PDGFBB, and Glutamax were obtained from LifeTechnologies (Grand Island, N.Y.). Para-toluene sulfonic acid, ammoniumhydroxide, IR780 and monocrotaline (C2401 Lot 031M1921V andLotSLBB7802V) were obtained from Sigma Aldrich (St. Louis, Mo.).Anti-phospho-AKT(Ser 473), anti-phospho-AKT (Thr308), pan-AKT (CST2920mouse mAB, and CST2965 rabbit mAb), anti-phospho-ERK1/2, antiphospho-STAT3, and total STAT3 antibodies were obtained from CellSignaling Technologies, (Waltham, Mass.). Anti-total ERK1/2 antibody wasobtained from Protein Simple (CA). Anti von-Willebrand Factor, actin,phospho-PDGFRα (Y754), and PDGFBB antibodies were obtained from AbCam(Cambridge, Mass.). Antibodies against PDGFAA (sc-128), PDGFR-alpha(sc-338), PDGFR-beta (sc-432) and p-PDGFR-beta (Tyr 1021) (sc-12909)were obtained from Santa Cruz Biotechnology (CA). 680LT goat anti-mouseIgG, IRDye 800 W goat anti-rabbit IgG, and Odyssey blocking buffer wereobtained from Licor (Lincoln, Nebr.).

In vitro kinase assay. A Z-lyte kinase assay was performed to determinethe inhibition of PDGFRalpha and PDGFRbeta mediated phosphorylation byPK10453 (Structure 2). Ten point titration curves were modeled tocalculate the IC50 (Invitrogen Select Screen®).

PASMC Proliferation Assay.

Human pulmonary artery smooth muscle cells (PASMC) were obtained fromCell Applications (San Diego, Calif.) and grown to 50% confluence in a96 well format. The cells were switched to serum free media 24 hoursprior to stimulation with PDGFBB 50 ng/ml and varying concentrations ofPK10453 (Structure 2). After 24 hours of treatment, a Cyquant NF Cellproliferation assay was performed (Invitrogen®), and the fluorescentsignal was measured with a Cytofluor Plate reader. Data is based on anaverage of 8 replicates at each concentration.

In cell Western (ICW). To compare the inhibitory profiles of PK10453(Structure 2) and imatinib for PDGFBB and PDGFAA stimulated AKTphosphorylation, ICWs were performed, with modifications, according tothe method of Chen et al., “A cell-based immunocytochemical assay formonitoring kinase signaling pathways and drug efficacy.” Analyticalbiochemistry; Vol. 338:136-42 (2005). HLFs were maintained in subcultureat no more than 6 passages in DMEM with 5% FBS and 4 mM Glutamax at 37°C., 5% CO₂. HLFs were plated and grown to 70-80% confluence in 96 wellplates then serum-starved for 48 hours. Cells were treated with drug(PK10453 or imatinib) at indicated concentrations for 30 min thenexposed to 10 ng/ml PDGF AA or BB for 7.5 min. Cells were fixed in 3.7%formaldehyde, washed with 0.1% Triton X-100, and treated with OdysseyBlocking Buffer for 90 min. Proteins were incubated overnight 1:100diluted rabbit mAb to phosphorylated AKT (Ser 473 or Thr 308) and 1:100mouse mAb to total Akt-pan-4040D. Antibodies were detected using IRDye680LT goat anti-mouse IgG and IRDye 800W goat α-rabbit IgG conjugatedantibodies. After washing, the signal was quantified using an OdysseyInfrared Imaging System (LI-COR). Phosphoprotein signal (800 nm) wasnormalized to total protein signal (700 nm) acquired from each well andexperimental duplicates on same plate were averaged and reported.

Animals.

Male Sprague Dawley rats (weight 320-330 grams; Taconic Inc.) were usedfor this study. Animals were housed in standard rat cages with a 12 hlight/dark cycle, and standard rat chow and water were provided adlibitum. Animals were cared for and used in accordance with NIHguidelines. All animal protocols were approved by the Bassett MedicalCenter and Pulmokine IACUC.

Formulation and Aerosol Delivery.

PK10453 (Structure 2) was dissolved at a concentration of 20 mg/ml in 1Mtosylic acid. Nebulization was performed with a PARI Nebulizer with anair pressure of 12.5 psi. The aerosol droplets were neutralized byammonia vapor that was passed into the aerosol air stream. The particleswere then dried by flowing through an annular ring of silica beadcartridges prior to reaching the exposure chamber. The 6-port exposurechamber was a nose-only exposure system custom designed and built byPowerscope Inc. (Minneapolis, Minn.). The vacuum flow rate at each portwas separately controlled by a flow meter. The aerosol particle size wasmeasured at the exit port of the drying column with an Anderson (MarkII) cascade impactor. The mass median aerodynamic diameter (MMAD) was 2μm and the associated geometric standard deviation (GSD) was 1.6.Imatinib mesylate was dissolved in water at 20 mg/ml and delivered by aPARI nebulizer then dried by passage through an annular ring of silicabead cartridges prior to inhalation.

Estimation of inhaled Dose.

Filters exposed to PK10453 (Structure 2) for either 4 or 8 min (n=6 eachgroup) via the Powerscope exposure chamber were placed in amber glassvials. Twelve milliliters of 1:3 (v/v) methanol:acetonitrile were addedto each vial containing a filter for approximately 1 hr, with periodicmixing, followed by sonication for 60 seconds. An aliquot was thendiluted 100-fold by adding 10 μL of unknown filter extract to 990 μL of1:3 (v/v) methanol:acetonitrile. Samples were vortex mixed for 30seconds, and then a 100 μL diluted aliquot was combined with 100 μL of172 ng/mL of a nonchemically related internal standard (PK18855) in 1:1methanol:water, vortex mixed and transferred to autosampler vials forLC-MS/MS analysis. Filter extracts were compared against a calibrationcurve prepared in 100% methanol (PharmOptima®, Inc.). The aerosolconcentration of PK10453 (Structure 2) in p/liter of air was calculatedbased on the average total μ of PK10453 (Structure 2) on the filters forthe 4 and 8 min exposure times, and the flow rate past each filter (0.8L/min). The inhaled dose was calculated with the average concentrationof PK10453/cm² filter paper (average of 4 and 8 min exposures), theaverage min ventilation measured by plethysmography (0.15 L/min), and anestimated deposition fraction of 0.1. The imatinib 8 min dose was basedon gravimetric analysis.

Imaging.

The spatial distribution of inhaled PK10453 (Structure 2) in the lungwas evaluated by fluorescent imaging. A near IR fluorescent tracer,IR-780, was added to the drug solution in the nebulizer to ensure driedaerosol particles contained both the drug and IR tracer. After a two minexposure, animals were placed under general anesthesia underwentintubation via tracheostomy, and the lungs were excised. OCT/PBS wasinfused via the pulmonary artery, the lung insufflated with air, and thelungs frozen in the vapor phase of liquid nitrogen. Serial approximate 2mm sections of lung were imaged on a Licor Odyssey Imager.

Pharmacokinetic Studies.

PK10453 (Structure 2) was administered intravenously or by inhalation toanimals, which were then euthanized at time 0, 10, 20, and 60 min (n=3each time point). Blood samples were taken by cardiac puncture, and thelungs excised. The lungs were homogenized and PK10453 (Structure 2)extracted with a 1:3 mixture of acetonitrile:methanol. Similarly, plasmawas extracted with a 1:3 mixture of acetonitrile:methanol. Drug wasassayed by LC MS/MS (PharmOptima Inc., Portage Mich.). First orderexponential curves were fit to the data with Excel. AUC was determinedwith the trapezoidal method of integration.

Efficacy study in the rat MCT model—PK10453 (Structure 2) dose responsestudy in the rat MCT Model. Male Sprague Dawley rats received MCT 60mg/kg IPMCT, and after 3 weeks, PK10453 (Structure 2) or vehicle controlwere administered by inhalation. Four groups were studied: vehiclecontrol (4 min exposure) and three treatment groups of PK10453(Structure 2) with exposure times 2 min (D2), 4 min (D4), or 8 min (D8)three times a day. These regimens were administered for two weeks. Thevehicle consisted of aerosolized 1M tosylic acid neutralized withammonia vapor as described above. The pH of a solution prepared bydissolving captured aerosol particles in water was measured for everydose and was consistently in the range of 5.5-6.0. At the end of thestudy, the RV systolic pressure was measured, and the heart chambersdissected and weighed.

Efficacy study in the rat MCT model—PK10453 (Structure 2) vs. imatinibin the rat MCT model. Male Sprague Dawley rats were given MCT 60 mg/kgIP. Three weeks later vehicle (1M tosylic acid), PK10453 (Structure 2 at20 mg/ml free base in 1 M tosylic acid), or imatinib mesylate (20 mg/mlin nebulizer solution) were administered to designated groups for 8 mininhalation exposures, three time a day, for two weeks. At the end of thestudy RVSP pressure was measured; lung and heart fixed in formalin. Formeasurement of RVSP animals were sedated with isoflurane, intubated viaa tracheostomy, and ventilated with a TOPOVENT pressure regulatedventilator (peak inspiratory pressure 18 cm H₂O, PEEP 5 cm). Aftersternotomy, a Scisense high fidelity catheter inserted via the RV apex.

Efficacy Study in the Rat MCT+PN Model.

Pneumonectomy and implantation of a TRM53P telemetry monitor in thepulmonary artery (Telemetry Research, New Zealand and ADInstruments,Colorado) was carried out in rats. Two weeks after MCT, PK10453(Structure 2) was administered three times daily for 1 week. Dosing wasbegun 2 weeks after MCT rather than 3 weeks, because in this moreaggressive model the animals developed PAH more quickly and developeddistress sooner than in MCT only treated animals (data not shown). Thetwo groups underwent 4 min exposures of either the vehicle control orPK10453 (Structure 2). Sampling of PA pressure was performed 5 minbefore each morning dose in ambulatory animals in room air (estimatedatmospheric pressure 716 mm Hg based on elevation of animal facility).In protocol 4 (imatinib vs. vehicle), the animals received DSI PAC40transmitters followed by monocrotaline 50 mg/kg IP (Lot SLBB7802V). Alower dose of MCT was used for this study, because attempts to use 60mg/kg of this lot of MCT resulted in the need for early euthanasia in ahigh proportion of animals due to weight loss and tachypnea. Two weeksafter MCT IP injection, vehicle (mesylate 3 mg/ml) or imatinib mesylate20 mg/ml in nebulizer solution) was administered for 8 min exposuresthree times a day for 9 days. Telemetry data was obtained for 10 mindaily before each morning dose for this protocol.

Measurement of PV Loops.

In a separate cohort of animals, the MCT+PN model was developed asdescribed above, and PK10453 (Structure 2) was then administered for 4or 8 min three times a day to the drug treated group. The vehiclecontrol group underwent 4 min exposures three times a day. PressureVolume (PV) loops were obtained with an admittance system (Scisense,Inc.) after 14 days of treatment, while rats were under generalanesthesia with isoflurane and 100% FiO₂. Also, or in the alternative,RV pressures were obtained in each group after 14 days of treatment. Ina subset of each group, pressure Volume (PV) loops were obtained with anadmittance system (high fidelity catheter FTE1918B, Scisense, Inc.)after 14 days of treatment. After induction of general anesthesia andintubation via tracheostomy, the rats were placed on a pressurecontrolled ventilator (TOPOVENT). General anesthesia consisted ofisoflurane and 100% FiO₂ with peak inspiratory pressure set at 18 cm,and PEEP 5 cm H₂O. A left thoracotomy was performed with admittancecatheter in the RV via the RV outflow tract.

Systemic Blood Pressure Study.

The effect of PK10453 (Structure 2) on systemic BP was studied inambulatory MCT treated rats with DSI PAC40 transmitters implanted in thedescending aorta. Three weeks after administration of MCT 60 mg/kg IP,animals inhaled PK10453 (Structure 2) or vehicle 3×/d with 4 minexposure for 7 days. Blood pressure was recorded before each morningdose.

Plethysmography.

Plethysmography was performed with an EMKA dual chamber plethysmographand IOX software. Parameters measured included breathing frequency,tidal volume, minute ventilation, peak inspiratory and expiratory flow,and airway resistance (SRaw). Animals were acclimatized to theplethysmograph for three days prior to first data acquisition.Measurements were made prior to the first dose of drug and at the end ofthe study.

Histology and Morphometric Analysis.

At the end of the study, the heart and lungs were removed fromventilated animals under general anesthesia. Heparinized saline wasinfused under pressure through the main pulmonary artery. The rightupper lobe was immediately tied off and placed in liquid nitrogen forWestern blot and NanoPro™ 100 assay analysis. The heart was removed, andthe RV free wall, interventricular septum and LV free wall dissected andweighed. Buffered formalin (10%) was infused under pressure both throughthe pulmonary artery and the trachea. Morphometric analysis wasperformed on H&E stained formalin fixed tissue sectioned at 8 μm. Themedia area and lumen area of pulmonary arterioles were measured withImage J software by a technician blinded to treatment group.Measurements were made on 20 pulmonary arterioles per section. The ratioof the lumen area to the total media area was determined. This rationormalizes the variation in total pulmonary arteriole area. In addition,occlusive analysis was performed in the monocrotaline plus pneumonectomystudy (specifically efficacy study 5) according to the method of Hommaet al., “Involvement of RhoA/Rho kinase signaling in protection againstmonocrotaline-induced pulmonary hypertension in pneumonectomized rats bydehydroepiandrosterone.” Am J Physiol Lung Cell Mol Physiol. Vol.295:L71-8 (2008). Briefly, pre-capillary arterioles were assigned grade0 for no evidence of neointimal lesions, grade 1 for less than 50%luminal occlusion, and grade 2 for greater than 50% occlusion. MassonTrichrome stains were performed on lung sections from the MCT+PN model.

NanoPro™ Immunoassay.

Relative differences in phosphorylated ERK1/2 and STAT isoforms weremeasured with a NanoPro100™ immunoassay system (Protein Simple/CellBiosciences, CA). See Fan et al., “Nanofluidic proteomic assay forserial analysis of oncoprotein activation in clinical specimens.” NatMed 15:566-571 (2009).

Immunohistochemistry.

Antigen retrieval was performed with citrate buffer (pH 6.0) orTris-EDTA buffer (pH 9.0). Immunohistochemistry was performed for thefollowing targets: CD20 (a B cell marker), CD3 (a T cell marker), vonWillebrand Factor (vWF), total STAT3, phosphoSTAT3 (Tyr705), totalPDGFR-alpha, total PDGFR-beta, and phosphoPDGFR-beta. Competing peptideswere available for PDGFR-alpha and phospho-PDGFR-beta. Signal detectionwas performed with an EXPOSE HRP/DAB kit (Abeam®).

Statistical Analysis.

Data are presented as mean±SEM unless otherwise noted. The GeneralLinear Model with the Bonferroni correction for multiple groupcomparisons was used (SPSS 14.0). Significance was set at the p=0.05level.

Example 1—Characterization of PK10453 (Structure 2)

FIGS. 1A-1D show graphs depicting IC₅₀ concentrations for Imatinib andPK10453 (Structure 2). An in vitro kinase assay demonstrated the IC₅₀for PK10453 at ATP K_(m) was 35 nM for PDGFR-α and 10.1 nM for thePDGFR-β. For imatinib the IC₅₀ at ATP K_(m) was 71 nM for PDGFR-α and607 nM for PDGFR-beta. FIGS. 2A-2E show graphs of and images of In CellWestern (ICW) assays demonstrating the lower IC₅₀ of PK10453 (Structure2) against PDGFAA and PDGFBB stimulated phosphorylation of AKT at Ser473and Thr308 compared to Imatinib in human fetal lung fibroblasts. TheIC₅₀ of PK10453 for PDGFBB stimulated AKT phosphorylation at Ser473 was0.13 μM compared to 1.8 μM for imatinib (p<0.01). The IC₅₀ of PK10453for PDGFBB stimulated AKT phosphorylation at Thr308 was 0.43 μM vs. 3.25μM for imatinib (p<0.001). The IC₅₀ concentrations of PK10453 andimatinib for PDGFAA stimulated phosphorylation of AKT were notsignificantly different.

Estimated Inhaled Dose—PK10453 (Structure 2) and Imatinib.

The average concentration of PK10453 was 62.4±3.3 μg/cm² filter paperfor the 4 min exposure, and 137±7.0 μg/cm² for the 8 min exposure, whichresulted in an aerosol concentration of 91.65 μg/L air for the 4 minexposure and 100.6 μg/L air for the 8 min exposure. The aerosolconcentration of imatinib based on gravimetric analysis was 167 μg/L.The average inhaled dose (8 min), assuming a deposition fraction of 0.1and rat weight 300 g, was approximately 20 μg/kg for PK10453, and 40μg/kg for imatinib, as shown in Table 1. The estimated inhaled dose wascalculated from the measured concentration of PK10453 (Structure 2) andgravimetric analysis of imatinib in the aerosol, the measured minuteventilation (MV), the estimated deposition fraction of 0.1, and ratweight 300 g.

TABLE 1 Total Lung Lung Aerosol Exposure MV MV*Exposure Depositiondeposition Total Total Lung deposited API Conc ug/L Min L/min timefraction fraction Inhaled ug Deposited ug deposited ug ug/kg PK1045396.13 8.00 0.15 1.20 0.10 0.60 115.36 11.54 6.92 23.07 Imatinib 167.48.00 0.15 1.20 0.10 0.60 200.88 20.09 12.05 40.18

Lung Distribution and Pharmacokinetics of Inhaled PK10453 (Structure 2).

Fluorescent images of the lung sections following inhalation of PK10453with IR780 tracer are shown in FIG. 3, where the flurorescence intensityis shown to be well distributed throughout the lungs. The network ofdarker lines arises from the connective tissue and therefore does notrepresent the airways affected by the disease. The spatial distributionof imatinib was similar (data not shown).

For the pharmacokinetic study, the concentration of PK10453 (Structure2) in lung when administered by inhalation was compared to theconcentration achieved with IV administration. As described in Morén“Aerosols in medicine: principles, diagnosis, and therapy.” Amsterdam;New York: Elsevier. (1993) and Phalen et al., “Inhalation exposuremethodology.” Environ Health Perspect 56:23-34 (1984)), it is possibleto estimate the pharmacokinetic advantage of inhalation relative tointravenous administration, R_(d), by comparing the AUC of a plot of thedrug concentration as a function of time following respiratory and IVadministration:R_(d)=[(AUC_(lung)/AUC_(plasma))respiratory]/[(AUC_(lung)/AUC_(plasma))IV]

The pharmacokinetic data was modeled to a first order exponential curve,and the AUC calculated from the curves (see Table 2). FIGS. 4A and 4Bshow the drug levels in lung and plasma as a function of time followinginhalation (INH) or intravenous (IV) administration of PK10453(Structure 2). The data indicate a 45 fold advantage of inhaled comparedto IV administered PK10453 (R_(d)=44.6).

TABLE 2 Y = AEXP (−bX) A (ng/g lung) b (min−1) R2 Lung (INH) 2468 0.030.89 Plasma (INH) 132.7 0.07 0.93 Lung (IV) 446 0.06 0.96 Plasma (IV)1266 0.07 0.92 AUC Lung (INH) 1001.82 Plasma (INH) 65.47 Lung (IV)211.89 Plasma (IV) 617.25 Rd 44.58

Example 2—MCT Model Efficacy

FIGS. 5A-5D depict the effect of PK10453 (Structure 2) on rightventricle (RV) systolic pressure (SP) and RV hypertrophy in the MCT andMCT+PN model systems. RVSP values are shown in FIG. 5A. In the vehiclegroup (n=6), RVSP was 80.4±2.6 mm Hg. For the treatment groups, D2(n=6), 51.4±6.5; D4 (n=6), 44.4±3.8; and D8 (n=5), 37.1±4.5 mm Hg(p<0.001). Normal control group RVSP was 28.5±2.6 mm Hg (n=3). In the D4group, there was a 44% reduction in RVSP, and in the D8 group, there wasa 54% reduction in RVSP compared to the vehicle treated group. There wasalso a significant reduction in the degree of RV hypertrophy as measuredby the ratio (RV+IVS)/LV weight. See FIG. 5B. The data are representedby this ratio because the septum is shared by the RV and LV. However,use of the RV/(IVS+LV) ratio also showed similar results.

Moreover, there were 6 animals in the vehicle group but accurate RV endsystolic pressures were not obtained due to bleeding in 2 animals.Therefore RV systolic pressure is based on n=4 in the vehicle group andwas 57.9±7.6 mm Hg. In the PK10453 (Structure 2) group (n=12) RV endsystolic pressure was 36.3±2.6 mm Hg, and in the imatinib group (n=6)was 31.8±1.8 mm Hg (p=0.001 Vehicle vs. PK10453; p=0.002 Vehicle vs.Imatinib, FIG. 5C). End systolic volume was greater in the vehicle group(158±12.6 μl) vs. PK10453 (99.5±10 μl) and imatinib (81±4.3 μl) (p=0.05vehicle vs. PK10453; p=0.014 vehicle vs. imatinib; p=NS PK10453 vs.imatinib). There were no significant differences between the groups forthe following parameters: end diastolic volume, ejection fraction,cardiac output, stroke work. The lumen to media ratio was improved byboth PK10453 and imatinib compared to vehicle in the MCT model (Vehicle(V, n=4): 0.55±0.1; PK10453 (D8, n=12): 0.94±0.08; Imatinib (18, n=5):0.99±0.07; p<0.01 D8 vs. V, p<0.05 18 vs. V, FIG. 5D).

Example 3—Efficacy Studies in the Rat MCT+PN Model

Telemetry Studies.

The results of the telemetry study in the rat MCT+PN model aredescribed. At day 0 prior to start of treatment, the PA systolicpressure in the vehicle groups was 41.0±11.7 mm Hg, and in the PK10453(Structure 2) group, was 43.1±3.5 mm Hg (p=NS). After five days oftreatment, the PA systolic pressure was 69.4±12.9 mm Hg in the vehiclegroup and was significantly lower at 47.3±3.0 mm Hg in the PK10453 group(p<0.01). On treatment day 8, the PA systolic pressure in the vehiclegroup was 83.5±8.5, but significantly lower at 47.3±4.9 mm Hg in thePK10453 group (p<0.001).

FIGS. 6A and 6B show graphs for telemetry studies in the rat MCT+PNmodel. In a separate PK10453 (Structure 2) telemetry study, at day 1prior to start of treatment, the PA systolic pressure in the vehiclegroup was 47.4±10.2 mm Hg, and in the PK10453 group, was 43.1±3.5 mm Hg(p=NS). After five days of treatment, the PA systolic pressure was67.4±11.4 mm Hg in the vehicle group and was significantly lower at47.2±3.0 mm Hg in the PK10453 group (p=0.03). On treatment day 9, the PAsystolic pressure in the vehicle group was 92.8±9.1 mm Hg, butsignificantly lower at 50.5±7 mm Hg in the PK10453 group (p=0.03). Forthe imatinib telemetry study (study 4), at dayl, the PA systolicpressure in the vehicle group was 51.4±8.9 mm Hg, and in the imatinibgroup 41.5±3.5 mm Hg. At treatment day 9 the PA systolic pressure in thevehicle group was 80.4±14.2 mm Hg, and in the imatinib group was 75.1±7mm Hg (p=NS).

Measurement of RV Pressure and PV Loops in the MCT+PN Model; PK10453(Structure 2) Dose Response Study.

In a separate cohort of animals, the MCT+PN model was developed asdescribed. FIGS. 7A-7D represent graphs relating to hemodynamic andmorphometric analyses in the rat MCT+PN model. RV pressure was obtainedafter 14 days of vehicle exposure, and PK10453 treatment with 4 min (D4)and 8 min exposures (D8) three times a day. In the vehicle group (n=9),RV systolic pressure was 75.7±7.1 mm Hg, in the D4 group (n=10) RVsystolic pressure was 40.4±2.7 mm Hg, and in the D8 MCT+PN group RVsystolic pressure was 43±3.0 mm Hg (p<0.001 V vs. D4 and V vs. D8; FIG.7A). PV loops were obtained in a subset of animals from each group(Vehicle n=3; D4 n=5, D8 n=4).

Example 4—MCT+PNMCT+PN Model Efficacy

PV Loop Study.

The RV end systolic pressure (ESP) was lower and the RV ejectionfraction (EF) was higher in both the D4 and D8 treatment groups comparedto vehicle control. Cardiac output in the D8 group was increasedcompared to the Vehicle group. See Table 3. The study animals underwentleft pneumonectomy followed 7 days later by MCT 60 mg/kg IP. Two weeksafter MCT administration, PK10453 (Structure 2) or vehicle were given byinhalation three times a day for two weeks. PV loops were acquired atthe end of this period. With respect to Table 3: V=vehicle; D4=4 mininhalation PK10453; D8=8 min inhalation PK10453; n=4 each group;*p<0.001; **p<0.01; § p<0.05 vs. V.

TABLE 3 Group Set HR (bpm) ESP (mm Hg) EDP (mm Hg) ESV (μl) EDV (μl) 5 V(μl) CO ml/min EF SW V Mean 290 83.21 10.31 487.17 621.32 137.15 39.0325.43 10123 SEM 25 3.49 1.24 148.32 139.49 14.19 0.62 8.36 2698 D4 Mean288 43.20* 2.62§ 144.14 408.95 264.81 77.59 65.4* 9818 SEM 21 6.08 0.3025.89 34.94 12.66 2.59 3.47 769 D8 Mean 315 38.44** 4.87 155.40 488.68333.28** 105.1** 67.1* 5481 SEM 41 1.43 1.86 22.69 52.00 49.81 15.514.59 1829

Effect of PK10453 (Structure 2) on RV Hypertrophy.

Treatment with PK10453 resulted in a significant decrease in RVhypertrophy in the rat MCT+PNMCT+PN model. See FIG. 7B. The (RV+IVS)/LVratio in the vehicle group (n=11) was 0.88±0.05, in the PK10453 D4 group(n=13) was 0.62±0.04, and in the PK10453 D8 group (n=7) was 0.68±0.05(p<0.001 D4 vs. V; p=0.012 D8 vs. V).

Analysis of Pulmonary Arteriole Histology and Morphology.

The lumen area to media area ratio (L/M) was significantly higher in thePK10453 (Structure 2) treated D8 group compared to the D4 or vehiclegroups: D8 (n=5) L/M 0.72±0.05, D4 (n=6) L/M 0.33±0.06, and the vehiclecontrol V (n=6): 0.26±0.04 (p<0.0001 D8 vs. V or D8 vs. D4). See FIG.7C. Occlusion analysis was performed on the same animal samples used forthe lumen/media ratio measurements. The occlusion analysis demonstrateda significant reduction in Grade 2 occlusion lesions in the PK10453 D8treatment group (V (n=6) 41.5±7.1%, D4 (n=6) 28.5±4.2%; D8 11.4±4.1%;p<0.01 D8 vs. V; see FIG. 7D.

FIGS. 8A-8D illustrate the effect of PK10453 (Structure 2) on neointimallesions in the rat MCT+PN model via 40× microscopic images of pulmonaryarteriole hypertrophy and intraluminal cellular proliferation of PK10453treated specimens. FIG. 8A shows an H&E stain of an occlusive (Grade 2)lesion in a vehicle treated animal (MCT+PN model); comparison is made toa Grade 0 vessel from a PK10453 (D8) treated animal. See FIG. 8B. Anexample of a Grade 2 lesion stained for phospho-PDGFRbeta is shown inFIG. 8C with comparison to a Grade 0 lesion from a PK10453 (D8) treatedanimal (MCT+PN model) in FIG. 8D. Staining for phosphoPDGFRbeta showedintense signal in a cobblestone pattern in Grade 2 lesions.

Further examples of pulmonary arteriole hypertrophy and intraluminalcellular proliferative lesions are shown as described, while thequantitative analysis is represented in FIG. 9. FIGS. 10A and 10B depictan immunohistochemical evaluation of MCT+PN samples. The lumen area tomedia area ratio (L/M) was significantly higher in the PK10453 treatedgroups compared to vehicle, where the higher dose, D8 (n=4) L/M1.17±0.07, the lower dose, D4 L/M 0.75±0.14, and the vehicle control V(n=6): 0.36±0.09 (p=0.032 D4 vs. V; p=0.00014 D8 vs. V; p=0.028 D8 vs.D4). The endothelial cell marker, vWF, showed signal predominantlywithin the pulmonary arterioles. The tyrosine705 phosphoSTAT3 antibodyshowed localization of pSTAT3 to nuclei of endothelial cells andperivascular cells. See FIG. 10A; and FIG. 10B (with PK10453 treatment).

Trichrome and Immunohistochemistry for Alpha-SMC Actin, and vWf.

The endothelial cell marker, vWF, showed signal predominantly within thepulmonary arterioles. And immuno-histochemistry for vascular SMCs (αSMCactin), endothelial cell markers (vWF) and trichrome stains of pulmonaryarterioles in the rat MCT+PN was performed to further characterize Grade0, 1, and 2 lesions. Grade 0 lesions were characterized by earlyneointimal (intraluminal) proliferation of endothelial cells (ECs) withpreservation of vascular SMCs in the media; Grade 1-2 lesions, byneointimal (intraluminal) proliferation/invasion of mixedmyofibroblast-like cells (MFs) and ECs with partial loss of vascularsmooth muscle cells in the medial layer; and advanced Grade 2 lesions,by extensive MF/EC intraluminal proliferation with complete loss of VSMcells in the medial layer and fibrotic replacement of the media.

FIGS. 11A-11I relate to vehicle treated animals (MCT+PN model) at 40×for immunohisto-chemically stained αSMC actin, Trichrome and vWF stains,which showed a mixed population of endothelial and myofibroblast cellscomprising the neointimal and proliferative lesions in pulmonaryarterioles in Grade 0, 1, and 2 lesions: Grade 0 lesions werecharacterized by early intraluminal endothelial cell proliferation, andpresence of vascular smooth muscle cells in the media (FIG. 11A, αSMCstain; FIG. 11D, trichrome; FIG. 1G, vWF). Grade 1-2 lesions hadextensive intraluminal myofibroblast-like cells, some endothelial cells,and partial fibrosis of medial layer (FIG. 11B, αSMC; FIG. 11E,trichrome; FIG. 11H. vWF). Advanced Grade 2 lesions were characterizedby extensive intraluminal myofibroblast-like and endothelial cellproliferation and complete fibrotic replacement of medial layer (FIG.11C, αSMC; FIG. 11F, trichrome; FIG. 11I. vWF). The long arrow points tothe intraluminal space with proliferative lesions, and the short arrowpoints to the medial layer of the pulmonary arterioles.

Example 5—Immunohistochemistry for PDGF Signaling

In pre-capillary pulmonary arterioles signaling through the PDGFR-βpathway was dominant. Signal for PDGFAA ligand and PDGFR-α were presentbut qualitatively lower than signal for PDGFBB and PDGFR-β.Phosphorylated PDGFR-β (pPDGFR-β) had a cobblestone appearance inneointimal cells and in perivascular cells and was stronger than signalfor phospho-PDGFR-α (PDGFR-α) in precapillary pulmonary arterioles.Minimal signal for pPDGFR-β or alpha was detected in the medial layersof the pre-capillary pulmonary arterioles.

FIGS. 12A-12F show 40×PDGFR signaling in the rat MCT+PN model. FIGS.12A-12F show PDGFAA in a pulmonary arteriole (A); PDGFBB (B); totalPDGFRα (C); total PDGFRPβ (D); phosphoPDGFRα (pPDGFRα; E); and pPDGFRPβ(F). Signal intensity was greater for PDGFBB, PDGFRPβ, and especiallypPDGFRPβ compared to PDGFAA, PDGFRα, and pPDGFRα. The pPDGFRPβ signalwas intense in a cobblestone pattern in neointimal proliferative andperivascular lesions. Signal intensity was relatively low in vesselmedia layer. Arrows point to vessel lumens with proliferative lesions(slides are from vehicle treatment).

In larger (>50 am) vessels, signal for pPDGFR-α was present in medialVSM cells. In contrast, pPDGFRPβ medial layer signal was low. FIGS.13A-13D show a comparison of pPDGFRα and pPDGFRPβ in larger pulmonaryarterioles using the rat MCT+PN model system.

Example 6—NanoPro™ Immunoassays and Western Blots

Nanopro™ Immunoassays for pAKT/AKT are shown in FIGS. 14A-14F andpSTAT3/STAT3 are shown in FIGS. 15A-15C. There was a significantreduction in the pSTAT3/STAT3 ratio in both the D4 and D8 groupscompared to vehicle. FIGS. 16A-16H show the results from experimentsusing the Nanopro™ immunoassay lumograms for phosphoERK1/2 (pERK1/2) andtotal ERK1/2 in the MCT+PN model. FIGS. 16A-16H show the effects ofinhaled PK10453 (Structure 2) on ppERK1/ERK1, pERK/ERK1, ppERK2/ERK2 andpERK2/ERK2 in lung homogenates. There were significant reductions inppERK1/ERK1 and pERK1/ERK1 in the D4 and D8 groups, respectively,compared to vehicle.

Example 7—PDGFAA Stimulates PDGFR-α, Whereas PDGFBB Binds & ActivatesPDGFR-β

FIGS. 17A-17D are graphic representations showing the effect ofimatinib, PK10453 (Structure 2), and PK10571 (Structure 2a) on PDGFAAvs. PDGFBB stimulated phosphorylation of ERK1 and ERK2 in human fetallung fibroblasts. The ratio diphosphorylated ERK1 to total ERK1(ppERK1/ERK1) was increased with PDGFAA or PDGFBB stimulation, andsignificantly decreased at 1 μM and 10 μMy concentration of imatinib,PK10453, and PK10571. The ratio diphosphorylated ERK2 to total ERK2(ppERK2/ERK2) was increased with PDGFAA or PDGFBB stimulation (10ng/ml), and significantly decreased at 1 uM and 10 uM concentration ofimatinib, PK10453, and PK10571. After PDGF BB stimulation, the ratio ofdiphosphorylated ERK1 to total ERK1 (ppERK1/ERK1) and diphosphorylatedERK2 to total ERK2 (ppERK2/ERK2) was more effectively decreased at 1 uMPK10453, and PK10571 compared to imatinib. Thus, PK10453 and PK10571 aremore potent inhibitors of PDGF BB stimulated ERK1 and ERK2phosphorylation compared to imatinib.

In particular, and with reference to FIGS. 17A-17D as noted above,PDGFAA and PDGF BB (10 ng/ml) stimulation of human fetal lungfibroblasts increased ppERK1/ERK1 and ppERK2/ERK compared to serum freemedia only controls (SF). Imatinib, PK10453 (Structure 2), and PK10571(Structure 2a) were equally effective at 1 uM in decreasing PDGF AAstimulated ppERK1 and ppERK2 formation (FIGS. 17A and 17C). However,PK10453 and PK10571 were more effective at 1 uM and 10 uM in decreasingPDGF BB stimulated ppERK1 and ppERK2 (FIGS. 17B and 17D). These datademonstrate that PK10453 and PK10571 are more effective in blockingsignal transduction through the PDGF receptor beta compared to imatinib.Data shown are mean±SEM. The differential effect of PK10453 and PK10571was more prominent in blocking ERK1 vs. ERK2 phosphorylation. At 1 uMimatinib had no effect on inhibition of ppERK1 formation whereas PK10453and PK10571 at 1 uM were effective in decreasing PDGFBB stimulatedppERK1 formation. PK10453=structure 2; PK10571=structure 2a. Plateletderived growth factor receptor alpha=PDGFR-alpha=PDGFR-α=PDGF receptoralpha=PDGF alpha receptor. Platelet derived growth factor receptorbeta=PDGFR-beta=PDGFR-β=PDGF receptor beta=PDGF beta receptor.

Example 8—PK10453 (Structure 2), PK10467 (Structure 3), PK10468(Structure 4), PK10569 (Structure 5) and PK10571 (Structure 2a) PossessLower IC₅₀ Concentrations Compared to Imatinib for Inhibiting PDGFBBStimulated AKT Phosphorylation in Fibroblasts

Fetal human lung fibroblasts grown in cell culture are used as a modelof fibroblast proliferation that occurs in pulmonary arterialhypertension, pulmonary fibrosis, and related disorders. FIGS. 18A-18Dare graphic representations of PK compounds: PK10453 (Structure 2),PK10467 (Structure 3), PK10468 (Structure 4), PK10569 (Structure 5) andPK10571 (Structure 2a), showing that all PK compounds possessed lowerIC₅₀ concentrations compared to imatinib for inhibiting PDGFBBstimulated AKT phosphorylation in fetal human lung fibroblasts. Thesedata highlight that PK10453, PK10467, PK10468, PK10569, and PK10571 aremore potent inhibitors of signal transduction mediated through the PDGFbeta receptor compared to imatinib. These data show the importance ofeffective inhibition of PDGF beta receptor signaling in addition to PDGFalpha receptor signaling as a treatment for pulmonary arterialhypertension, pulmonary fibrosis, and related disorders which can beachieved with PK10453, PK10467, PK10468, PK10569, and PK10571. As usedabove and throughout the application, the PK compounds and structuredesignations are used interchangeably, as follows: PK10453=Structure 2;PK10571=Structure 2a; PK10467=Structure 3; PK10468=Structure 4; andPK10569=Structure 5.

Example 9—Body Weights, Systemic BP, and Plethysmography Studies

Compared to vehicle, there was a trend to a slower rate of decline inbody weight in the treated vs. vehicle groups. See FIG. 19. On day sevenof treatment, systolic BP was 111±21 mmHg in the MCT vehicle group (n=3)compared to 131±10 mmHg in the MCT PK10453 group (n=3), as shown in FIG.20. Two-chamber plethysmography was measured at day 1 and day 15 ofPK10453/vehicle administration in the rat MCT+PNMCT+PN model. Theresults are shown in Table 4. Treatment with PK10453 was associated witha slower decline in minute ventilation (MV), and a significantimprovement in peak inspiratory flow (PIF) and peak expiratory flow(PEF) in the 4 min exposure group (D4) compared to vehicle.

TABLE 4 Day 1 TV MV Day 15 Drug Group PIF PEF (ml) (ml/min) f SRaw PIFPEF TV MV f SRaw V (n = 6) mean 8.81 9.68 0.86 193.66 244.79 40.37 4.975.86 0.52 107.59 214.43 38.93 sem 0.79 0.98 0.14 20.66 28.02 4.11 0.390.44 0.07 9.58 13.83 6.53 D4 (n = 5) mean 9.82 11.04 1.00 223.24 224.6839.73 7.82* 9.33* 0.85 176.12§ 217.12 33.09 sem 0.70 0.56 0.07 11.999.87 3.33 0.34 0.67 0.12 14.53 18.64 4.80 D8 (n = 5) mean 8.54 9.43 0.74174.68 259.13 36.01 6.06 6.64 0.63 128.49 232.11 49.26 sem 0.72 1.010.15 22.32 26.42 3.82 0.84 0.99 0.16 19.47 30.71 7.11 Abbreviations:PIF: peak inspiratory flow; PEF: peak expiratory flow; TV: tidal volume;MV: minute ventilation; f: breathing frequency (breaths per minute);SRaw: airway resistance *p < 0.01 D4 vs. V; §p = 0.02 D4 vs. V.

Example 10—Discussion and Applied Embodiments

The PDGF signaling pathway has been found to be activated in humanpulmonary arterial hypertension (PAH) and in animal models of thedisease. This study tested the hypothesis that a novel, non-selectiveinhaled PDGF receptor inhibitor, PK10453 (Structure 2), would decreasepulmonary hypertension both in the rat monocrotaline (MCT) model and therat MCT plus pneumonectomy (+PN) model of PAH. PK10453 delivered byinhalation, for four (D4) and eight (D8) min exposures three times a dayfor two weeks, decreased right ventricular systolic pressure (RVSP) inboth the rat MCT and rat MCT+PN models: vehicle MCT group (n=6) RVSP was80.4±2.6 mm Hg; in the D4 MCT group (n=6), 44.4±5.8 mm Hg; and in the D8MCT group (n=5), 37.1±4.5 mm Hg (p<0.001 vs. vehicle); in the vehicleMCT+PN group (n=9) RVSP was 75.7±7.1 mm Hg; in the D4 MCT+PN group(n=10), 40.4±2.7 mm Hg, and in the D8 MCT+PN group (n=8), 43.0±3.0 mm Hg(p<0.001). In the rat MCT+PN model, continuous telemetry monitoring ofpulmonary artery pressures also demonstrated that PK10453 prevented theprogression of PAH. Imatinib given by inhalation was equally effectivein the MCT model, but was not effective in the MCT+PN model.

Immunohistochemistry demonstrated increased activation of the PDGFPβreceptor compared to the PDGFα receptor in neointimal and perivascularlesions found in the MCT+PN model. It was shown that imatinib isselective for the PDGFα receptor whereas PK10453 has a lower IC₅₀ forinhibition of kinase activity of both the PDGFα and PDGFPβ receptorscompared to imatinib. PK10453 decreased the ratio of phosphorylated AKT(Ser473) to total AKT, phosphorylated STAT3 (Y705) to total STAT3, theratio of diphosphorylated ERK1 to total ERK1 and the ratio ofmonophosphorylated ERK1 to total ERK1 in lung extracts from MCT+PNanimals. In short, PK10453, when delivered by inhalation, significantlydecreased the progression of PAH in the rat MCT and MCT+PN models.Non-selective inhibition of both PDGFα and PDGFPβ receptors thereforehas a therapeutic advantage over the selective inhibition of PDGFRα atleast in PAH and related diseases.

Accordingly, and for the first time, it has been shown that a novel,non-selective PDGF receptor inhibitor, PK10453 (Structure 2), whenadministered by inhalation, decreased the severity of PAH in two animalmodels of the disease: the rat MCT, and the rat MCT+PN model. As such,because PK10453 is highly potent against both the PDGFRα and PDGFRPβreceptors, while imatinib is selective for the PDGFRα receptor, PK10453possesses surprisingly superior efficacy. Both PK10453 and imatinib wereeffective in the rat MCT model, but only PK10453 decreased pulmonaryhypertension in the rat MCT+PN model when administered by inhalation.One reason for this differential effect may be due to hyper-activationof signaling through the PDGFRPβ receptor in precapillary pulmonaryarteriole neointimal lesions compared to the PDGFRα receptor in the ratMCT+PN model.

Accordingly, the present data demonstrates that a novel, non-selective,PDGF receptor inhibitor PK10453 (Structure 2) when delivered byinhalation prevented the progression of PAH in both the rat MCT and therat MCT+PN models. Of note, this is the first study to report efficacyof PDGF receptor inhibition in the rat MCT+PN model. A sustainedreduction in PA pressure was also found in ambulatory PAH (MCT+PN)animals treated with PK10453. Concomitant with a significant reductionof PA and RV systolic pressure in these models, a reduction in RVhypertrophy and an improvement in the lumen to media ratio of pulmonaryarterioles were demonstrated. Pressure volume loops displayed animprovement in RV ejection fraction, a higher cardiac output, and atrend towards lower stroke work in PK10453 treated animals compared tocontrol animals. In lung extracts of PK10453 treated animals, there wasa significant reduction in the pAKT(Ser473)/AKT, pSTAT3/STAT3,ppERK1/ERK1 and pERK1/ERK1 ratios.

Because PAH is a disease substantially localized to the lung, thehypothesis was tested that direct administration of the drug to thetarget site via inhalation would offer the advantage of higher localconcentrations (greater efficacy) and lower systemic concentrations ofdrug (lower side effects). Pharmacokinetic studies demonstrated a 45fold advantage of inhalation delivery compared to intravenousadministration of PK10453 (Structure 2). While PK10453 decreased RVsystolic pressure by 50% in the rat MCT model, it did not have anadverse effect on systemic BP. Additionally, inhaled PK10453 did notadversely affect lung function over a 2-wk course.

In the rat MCT model the present inventor compared inhaled PK10453 toinhaled imatinib and found both to be equally effective. These resultsare consistent with prior reports that the PDGF receptor inhibitorimatinib, when delivered systemically, decreased pulmonary hypertensionin the rat MCT model. See Schermuly et al., “Reversal of experimentalpulmonary hypertension by PDGF inhibition.” J Clin Invest; 115:2811-21(2005). However, in the rat MCT+PN model while inhaled PK10453 waseffective in lowering pulmonary pressures, inhaled imatinib was not. Therat MCT+PN model is a more aggressive model of PAH compared to the MCTonly model, and may more accurately reflect the pathology of the humandisease. White et al., “Plexiform-like lesions and increased tissuefactor expression in a rat model of severe pulmonary arterialhypertension.” Am J Physiol Lung Cell Mol Physiol; 293:L583-90 (2007).In vitro measurement of IC₅₀ for inhibition of PDGF-α and -β receptorsshowed that PK10453 was more potent than imatinib against both isoforms,and that imatinib is only a modest inhibitor of the PDGFRPβ isoformImmunohistochemistry demonstrated that the neointimal lesions in the ratMCT+PN model have high levels of phospho PDGFRPβ, with less pPDGFRα.These findings explain why non-selective inhibition of both PDGFRPβ andPDGFRα provided a therapeutic advantage over the selective inhibition ofPDGFRα.

The present data are consistent with Panzhinskiy et al., “Hypoxiainduces unique proliferative response in adventitial fibroblasts byactivating PDGFbeta receptorm-JNK1 signaling.” Cardiovasc Res; 95:356-65(2012), for the neonatal calve model of high altitude induced pulmonaryhypertension. In that model extensive perivascular proliferation ofadventitial fibroblasts was demonstrated along with activation ofpPDGFRβ. These lesions are similar to the pattern observed in the ratMCT+PN model for the present studies. These findings are also consistentwith those reported for human PAH. Perros et al., “Platelet-derivedgrowth factor expression and function in idiopathic pulmonary arterialhypertension.” Am J Respir Crit CareMed; 178:81-8 (2008), describing thedistribution of PDGFA, PDGFB, PDGFRα, PDGFRPβ and pPDGFRPβ in pulmonaryarterial lesions of patients with PAH. PDGFRα expression was foundmainly within the muscular medial layer of hypertrophied pulmonaryarterioles, whereas PDGFR3 and pPDGFRβ were dominant in endothelialcells of plexiform lesions.

The selectivity of imatinib for PDGFRα has not been previouslyemphasized in studies of PAH. Inhibition by imatinib of PDGFAAstimulated PDGFRα phosphorylation was reported to be 0.1 μM; whereasinhibition of PDGFBB stimulated PDGFRPβ phosphorylation at 0.38 μM. See,e.g., Deininger et al., “The development of imatinib as a therapeuticagent for chronic myeloid leukemia.” Blood; 105:2640-53 (2005). Here,however, it was determined that, at [ATP]Km(app), imatinib was 10 foldmore selective for PDGFRα compared to the -beta receptor (IC₅₀ againstPDGFRα 71 nM vs. 607 nM for PDGFRPβ). Most PAH related cell basedstudies interrogating the PDGFR pathway employed high doses of imatinib(5-10 μM) and thus preclude distinction between PDGFRα and β receptorinhibition.

Wu et al., “Comprehensive dissection of PDGF-PDGFR signaling pathways inPDGFR genetically defined cells.” PLoS One; 3:e3794 (2008), examinedPDGFR signaling in genetically defined mouse embryonic fibroblasts(MEFs). The MEFs were engineered to express only the PDGFRα, PDGFRPβ,both or neither receptor. Signaling through the PDGFRα receptor and thePDGFRPβ receptor were found to have both shared and distinct pathways.Thirty-three gene sets were distinctly activated by PDGFRα and β byPDGFRPβ. PDGFRα/β heterodimers activated components of NFKκB and IL-6signaling. Calcium flux pathways were regulated by both PDGFRα andPDGFRβ. Signaling involved with angiogenesis was solely regulated viathe PDGFRPβ pathway. This finding comports with the selective increasein phosphoPDGFRPβ found with neointimal lesions of precapillarypulmonary arterioles using the MCT+PN model.

PDGFBB has been found to induce phosphorylation of AKT at Ser473 inpulmonary artery smooth muscle cells and fibroblasts, but not pulmonaryarterial endothelial cells. See Ogawa et al., “PDGF enhancesstore-operated Ca²⁺ entry by upregulating STIM1/Orail via activation ofAkt/mTOR in human pulmonary arterial smooth muscle cells.” Am J PhysiolCell Physiol; 302:C405-11 (2012). Increased phosphorylation of AKT(Ser473) was also found in cells with a smooth muscle phenotype fromendarterectomies of patients with chronic thromboembolic pulmonaryarterial hypertension. See Ogawa et al., “Inhibition of mTOR attenuatesstore-operated Ca²⁺ entry in cells from endarterectomized tissues ofpatients with chronic thromboembolic pulmonary hypertension.” Am JPhysiol Lung Cell Mol Physiol; 297:L666-76 (2009). PDGFBB stimulationincreased store operated calcium entry via the AKT/mTOR pathway in thesecells. See id.

In pulmonary artery smooth muscle cells from control and monocrotalinetreated rats, however, imatinib (0.1 μM) decreased fetal calf serumstimulated Ser473 AKT phosphorylation, but had no effect onphosphorylation of AKT at Thr30825. At this concentration it is likelythat imatinib was acting via the PDGFoc receptor. Wu et al. (2008) foundthat STI-571 (imatinib) at 5 μM blocked PDGFBB stimulated AKTphosphorylation (SER473) in both PDGFRPβ null and PDGFRα null celllines. The present invention included an ICW to examine PDGFAA andPDGFBB stimulation of AKT (Ser473) and AKT (Thr308) phosphorylation infetal human lung fibroblasts. Inhibition by imatinib was compared toPK10453 inhibition of PDGFAA or PDGFBB stimulated AKT phosphorylation,and found that PK10453 was more potent.

Nano-fluidic proteomic immunoassays, moreover, were employed to quantifyphosphorylated species of AKT, STAT3 and ERK1/2 in lung extracts ofMCT+PN animals. A significant reduction of phospho-AKT (Ser473),phospho-STAT3 and ppERK1/ERK and pERK1/ERK1 in the PK10453 treatedgroups was found as compared to vehicle. Schermuly et al. (2008)demonstrated a reduction in pERK1/2 by imatinib in the rat MCT model ofPAH. Jasmin et al., “Short-term administration of a cell-permeablecaveolin-1 peptide prevents the development of monocrotaline-inducedpulmonary hypertension and right ventricular hypertrophy.” Circulation;114:912-20 (2006), have shown activation of STAT3 in the rat MCT model,and Masri et al., “Hyperproliferative apoptosis-resistant endothelialcells in idiopathic pulmonary arterial hypertension.” Am J Physiol LungCell Mol Physiol; 293:L548-54 (2007), found that STAT3 was activated inhuman idiopathic PAH. The nanofluidic proteomic immunoassays of thepresent invention were previously used to examine the effects ofimatinib on pSTAT3, and pERK1/2 in chronic myelogenous leukemia (CML).See Fan et al., “Nanofluidic proteomic assay for serial analysis ofoncoprotein activation in clinical specimens.” Nature medicine;15:566-71 (2009). This assay has utility in distinguishingmonophosphorylated isoforms and diphosphorylated isoforms of proteins.For example, patients with CML who responded to imatinib had a distinctreduction in levels of monophosphorylated ERK214. Here, the ERK1 isoformand both the diphosphorylated form of ERK1 and the monophosphorylatedform of ERK1 predominated in lungs of MCT pneumonectomized rats.Treatment with PK10453 significantly decreased ppERK1/ERK andpERK1/ERK1.

Occlusion analyses were performed in accordance with the method of Hommaet al., “Involvement of RhoA/Rho kinase signaling in protection againstmonocrotaline-induced pulmonary hypertension in pneumonectomized rats bydehydroepiandrosterone.” Am J Physiol Lung Cell Mol Physiol; 295:L71-8(2008). In the rat MCT+PN model, the higher dose of inhaled PK10453 wasassociated with fewer Grade 2 occlusive lesions. These lesions were thencharacterized by immunohistochemistry with markers for vascular smoothmuscle cells, and endothelial cells, and performed trichrome stains todifferentiate muscular from fibrotic lesions. It was determined that theneointimal proliferative grade 1-2 lesions contained myofibroblasts andendothelial cells. In advanced grade 2 lesions there was fibroticreplacement of the vessel media. The origin of myofibroblasts in theselesions is not entirely clear. They could originate from infiltration ofperi-vascular fibroblasts or pericytes, from circulating stem cells,resident progenitor cells, or as a consequence ofendothelial-mesenchymal transition. See Yeager et al., “Progenitor cellsin pulmonary vascular remodeling.” Pulm Circ; 1:3-16 (2011). While theselesions were detected, it is reasonable to propose that the type 1lesion is an earlier stage lesion that can progress to type 2 and type3. In this model, intraluminal endothelial cells proliferate, transitionto a myofibroblast phenotype (and/or the lumen is infiltrated byperivascular cells/myofibroblasts) and progressively occlude the vessellumen.

Sakao et al., “Reversible or irreversible remodeling in pulmonaryarterial hypertension.” Am J Respir Cell Mol Biol; 43:629-34 (2010),have highlighted the importance of distinguishing regression of vascularmuscularization (reverse remodeling) from potentially irreversibleendothelial cell proliferation in PAH. The data presented here showsthat signaling through the PDGFRα pathway plays an important role invascular remodeling of larger pulmonary arterioles in PAH, whereas thePDGFRPβ pathway is more important in the proliferative neointimallesions of precapillary pulmonary arterioles. Targeting the PDGFRPβpathway with a PDGFR inhibitor that potently blocks this isoform (morepotently than imatinib) may influence progression of these lesions. Ifsuch lesions are therefore treated before full fibrotic replacement andvessel regression reversibility of these lesions may exist.

In conclusion, an inhaled, non-selective PDGF receptor inhibitor,PK10453 (Structure 2), was effective in both the MCT, and MCT+PN ratmodels of PAH. Treatment with PK10453 was associated with a significantreduction in pulmonary arterial pressures in ambulatory animals, animprovement in right ventricular function, and a reduction in RVhypertrophy. Histologic analysis demonstrated an improvement in thepulmonary arteriole lumen to media ratio in animals treated with PK10453and a decrease in the phosphorylation state of AKT (Ser473), STAT3 andERK1. There was no significant effect of PK10453 (Structure 2) onsystemic blood pressure, and no adverse effect of PK10453 on lungfunction. In contrast to imatinib, PK10453 is not selective for thePDGFRα receptor, but rather is highly potent against both the PDGFRα andβ isoforms. Because the PDGFRPβ pathway is more highly activated thanthe PDGFRα receptor in plexiform lesions of PAH, a non-selective PDGFRinhibitor, e.g., PK10453, thus possesses efficacy against PAH andrelated diseases and disease pathways.

What is claimed is:
 1. A method of reducing phosphorylation of anextracellular signal-regulated kinase (ERK), the method comprisingadministering to a subject a therapeutically-effective amount of acompound of the formula:

or a pharmaceutically-acceptable salt thereof.
 2. The method of claim 1,wherein the ERK is ERK1.
 3. The method of claim 1, wherein the ERK isERK2.
 4. The method of claim 1, wherein the phosphorylation isPDGFBB-stimulated phosphorylation.
 5. The method of claim 1, wherein thereduction in phosphorylation is measured in human lung fibroblasts. 6.The method of claim 1, wherein the reduction is observed at aconcentration of 1 μM of the compound.
 7. The method of claim 1, whereinthe reduction is observed at a concentration of 10 μM of the compound.8. The method of claim 1, wherein the administration is by inhalation.9. The method of claim 1, wherein the administration is oral.
 10. Themethod of claim 1, wherein the method inhibits a kinase receptor. 11.The method of claim 10, wherein the kinase receptor is a receptortyrosine kinase.
 12. The method of claim 11, wherein the receptortyrosine kinase is c-Kit.
 13. The method of claim 11, wherein thereceptor tyrosine kinase is PDGFR.
 14. A method of reducingphosphorylation of a serine/threonine kinase, the method comprisingadministering to a subject a therapeutically-effective amount of acompound of the formula:

or a pharmaceutically-acceptable salt thereof, wherein theserine/threonine kinase is AKT.
 15. The method of claim 14, wherein thephosphorylation is PDGFBB-stimulated phosphorylation.
 16. The method ofclaim 14, wherein the reduction in phosphorylation is measured in humanlung fibroblasts.
 17. The method of claim 14, wherein the reduction isobserved at a concentration of 1 μM of the compound.
 18. The method ofclaim 14, wherein the reduction is observed at a concentration of 10 μMof the compound.
 19. The method of claim 14, wherein the administrationis by inhalation.
 20. The method of claim 14, wherein the administrationis oral.